ABSTRACT A number of radioimmunoassay systems have been developed for quantitation of a variety of steroid hormones. Suitable antisera were obtained by immunizing rabbits with steroid derivatives conjugated at various positions to bovine serum albumin. The specificity of the antisera varied directly with the position of conjugation. Changes in the latter resulted in greater than 2000-fold shifts in relative inhibitory activity for a given steroid. The data indicate that conjugation should be done through a region (such as position 6) that is remote from distinguishing functional groups. The specificity data and nature of the inhibition curves suggest that the major portion of the steroid molecule is being recognized as a single immunoreactive unit. The advantages and problems associated with use of radioiodinated conjugates for labeled steroids are discussed. With conjugates, it has been possible to quantitate reliably as little as one pg of some steroids in a single assay tube.