An enzyme immunoassay for cortisol in human plasma using an antiserum raised against cortisol-3-O-carboxy-methyloxime bovine serum albumin and cortisol-21-hemisuccinate conjugated to penicillinase as tracer is described. Although employing immunoassay plates for separation of antigen-antibody complex from the free components was less time consuming, the slope and sensitivity of the standard curve were improved by the addition of goat anti-rabbit gamma globulin for precipitating the complex. There was good correlation between radioimmunoassay and enzyme immunoassay results obtained for cortisol levels present in normal human plasma.