Previous work has shown that the 21-hydroxylation of progesterone in the hepatic microsomal fraction of outbred NZW rabbits varies over a tenfold range. In contrast, the 16-hydroxylase activity is relatively constant and is not correlated to the activity of the 21-hydroxylase. The distribution of the 21-hydroxylase activity is roughly bimodal with about one-third of the animals (21-H) exhibiting 21-hydroxylase activity exceeding 1 nmol/min/mg microsomal protein, whereas the remainder (21-L) generally exhibit an activity that is less than 1 nmol/min/mg protein. To determine if this was due to a transient phenomenon, liver punch biopsies were collected from 28 rabbits at intervals of approximately three weeks for at least three serial samples. The 21- and 16-hydroxylase activities were determined in the postmitochondrial fractions of these biopsy samples. A substantial variability in both 21- and 16-hydroxylase activities was observed for serial biopsy samples from individual rabbits. The variation of the 16-hydroxylase activity paralleled, however, that of the 21-hydroxylase, thus suggesting that the variation between biopsy samples for individual rabbits was due to factors such as contamination with blood and connective tissue, which would affect both activities equally. Rabbits, therefore, were phenotyped as 21-H or 21-L on the basis of the 21/16-hydroxylase ratio. The mean ratio was 3.2 +/- 1.2 and 0.8 +/- 0.3 for rabbits phenotyped as 21-H and 21-L, respectively. Similar values for this ratio were obtained for the other group of rabbits phenotyped as 21-H and 21-L, 3.8 +/- 1.6 and 0.5 +/- 0.2, respectively, following the isolation of microsomes from whole liver homogenates. The ratio of 21/16-hydroxylase activity was found to be relatively constant for biopsy samples obtained from the same animal over the course of this study, thus indicating that the elevated 21-hydroxylase activity is not a transient phenomenon.