Absorbance changes in the spectrum of cytochrome P-450 were related to the inhibition of [26-14C]cholesterol oxidation to [14C]isocaproate and pregnenolone in mitochondria from bovine corpus luteum produced by two types of ligands. Nitrogenous inhibitors, such as aminoglutethimide, elicit an absorption maximum at about 427 nm and a minimum at about 393 nm (type II), while steroidal inhibitors, such as (20R)-20-(p-tolyl)-5-pregnene-3beta,20-diol (20-tolyl-pregnenediol), cause difference spectra with maximum at about 420 nm and minimum at about 390 nm (reverse type I). The magnitude of spectral change and the amount of inhibition of pregnenolone synthesis by aminoglutethimide are closely correlated at concentrations ranging from 5 to 750 muM and by the model steroid, 20-tolyl-pregnenediol, at concentrations from 0.5 to 25 muM. The responses are concentration dependent and linear over the range of effective concentrations. The concentrations of inhibitors for the half-maximal inhibition of pregnenolone biosynthesis are identical with the concentrations producing half-maximal spectral changes within experimental error. Displacement of substrate from cytochrome P-450 and/or stabilization of the redox potential subsequent to to the ligation of heme iron is proposed as the specific mechanism of cholesterol side chain cleavage inhibition. Finding, together, the two procedures offer a sensitive, specific, and accurate means of screening inhibitors of the cholesterol side chain cleavage system.