We describe a simple radioimmunoassay of plasma cortisol, which can be performed in 3 h and which requires no purification, heating, or refrigeration steps. The plasma proteins are inhibited through direct competition between them and the antiserum at room temperature, at which the antiserum's affinity exceeds that of the binding proteins. Plasma, diluted with water, is incubated for 3 h at room temperature with [3H]cortisol and the antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum was r = 0.954 (P less than 0.001), and the slope was 0.661. With three other antisera it was r = 0.922 (P less than 0.001) and slope 0.644. Plasmas with abnormal protein concentrations (i.e., from preganant women, and after corticotropin administration), tested to examine the validity of the method for routine use, and to define the role of the protein carriers, showed r = 0.859 (P less than 0.001) and slope 0.726 for the four antisera used. Additional samples, assayed with diluted standards plus stripped plasma, showed a correlation with the usual extraction method of r = 0.945 (P less than 0.001) and slope 1.026.