The steroid glucuronides estrone glucuronide and pregnanediol glucuronide were synthesised using the O-glycosylation reaction of a glycosyl donor with the appropriate steroid under standard Koenigs-Knorr conditions. X-ray crystallographic studies showed that the synthetic estrone glucuronide molecule had the correct stereochemistry. Estrone glucuronide and pregnanediol glucuronide conjugates of hen egg white lysozyme were prepared by both the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugate but the active ester procedure gave a more diverse range of products. Unreacted lysozyme, which was present in all cases, was removed by a combination of cation-exchange and hydrophobic-interaction chromatography to give purified conjugate material whose lytic activity was inhibited by over 90% in the presence of excess anti-steroid glucuronide antibody. Steroid glucuronide-lysozyme conjugates purified in this way could be used in a homogeneous enzyme immunoassay system to measure the levels of urinary estrone and pregnanediol glucuronide encountered in a normal menstrual cycle. Chromatography of the conjugation reaction mixtures on an S-Sepharose (fast flow) column in the presence of 7 M urea allowed the isolation of the different conjugate products. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride method gave one major derivative exclusively acylated at lysine residue 33 while acylation with pregnanediol glucuronide gave two major derivatives exclusively acylated at lysine residues 33 and 97 respectively. On the other hand, conjugation of lysozyme with the two steroid glucuronides by the active ester method gave six derivatives which were acylated at combinations of one or more of three lysine residues, 33, 97, and 116. The correlation of the protein environments of the lysine amino groups in the crystal structure of lysozyme with the acylation positions in the conjugate families suggested that these positions were determined not only by the surface accessibility and nucleophilicity of the lysine residues but also by the steroid glucuronide and the acylating reagent. Computer derived three dimensional structures of the estrone glucuronide- lysozyme and pregnanediol glucuronide-lysozyme conjugates suggested that the enzyme conjugate may be inactivated by the antibody in the immune complex by either providing a physical barrier to approach by the large bacterial substrate or by disrupting the binding of the bacterial cell wall polymer into the active site cleft. The lytic activity of the estrone glucuronide-lysozyme E3 conjugate was not inhibited in the presence of excess antibody when the small chitohexaose substrate was used, implying that the substrate could access the active site even when the conjugate was bound by antibody. The detailed characterisation of the mixed anhydride estrone glucuronide-lysozyme conjugate coupled with the current knowledge of the antigenic determinants of hen egg white lysozyme has made it possible to design, in principle, a novel sandwich solid phase immunoassay format for the measurement of estrone glucuronide levels.