The effects of the combination of antiserum and enzyme-labeled steroid and of the molar ratio of steroid to enzyme in the preparation of the conjugate on sensitivity and specificity in enzyme immunoassay of testosterone have been investigated. The enzyme labeling of testosterone was carried out by the N-succinimidyl ester method. Six testosterone derivatives were covalently linked to β-galactosidase at various molar ratios. The anti-testosterone antiserum used was that raised against the 4-O-hemiglutaroyl 4-hydroxytestosterone-bovine serum albumin conjugate. Immunological properties of the conjugate in both homologous and heterologous systems were examined. The number of steroid molecules incorporated per enzyme molecule markedly influenced the sensitivity in enzyme immunoassay. Cross-reactivity was also tested with 10 kinds of closely related steroids. The effect of site heterology on specificity was more significant than that of bridge heterology.