The production of estradiol antibodies by two immunization procedures was monitored by means of an enzyme-immunoassay. Procedure A consisted of three intramuscular injections given at two-week intervals, followed by five intravenous booster injections and procedure B consisted of multiple intradermal injections given once. Procedure A gave much higher antibody titer. In both procedures the sensitivities of assays using the antisera increased initially and reached a plateau after three to four months of immunization. Consistent changes in specificities were observed. A shortened procedure A is proposed as a simple and efficient procedure for raising steroid antibodies in rabbits.