The corticosteroid side-chain isomerases of mammalian liver catalyze the interconversion of the ketol and aldol side chains. In the mouse, isomerase was low in C57BL/6 (B6) mice (130 pmol/mg protein . 2 h) and high in BALB/c (C) mice (230 pmol/mg protein . 2 h). From analysis of hybrids between B6 and C and of backcrosses of these hybrids to B6, it was concluded that isomerase levels are controlled by a single autosomal gene dominant for high activity. The distribution of high and low isomerase levels in a series of CXB/By recombinant inbred strains of mice was consistent with linkage of the isomerase gene to H-2. Congenic BALB.B mice (H-2b haplotype from C57BL/10) had low isomerase activities corresponding to C57BL/10, not the high activity of the background strain BALB/c(H-2d). Similarly, BN10.D2 congenic mice (H-2d haplotype from the DBA/2 strain) had high activity characteristic of DBA/2. In the (C X B6)F1, (C X BALB.B)F1 and (B10 X B10.D2)F1 hybrids, all of which are H-2d/H-2d heterozygotes, isomerase activity was high. The association of isomerase levels with H-2 type was further confirmed in mice of the following backcrosses: (C X BALB.B)F1 X BALB.B, (C X B6)F1 X B6 and (B10 X B10.D2)F1 X B10. H-2b/H-2b homozygous segregants had consistently low activity and H-2b/H-2d heterozygous segregants had consistently high activity. It was concluded that the level of corticosteroid side-chain isomerase activity in mouse liver is controlled by a gene(s) in the region of the H-2 locus on chromosome 17.