Using in vitro and in vivo methods we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11ß -hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalysed by CYP11B2 (11ß -hydroxylation of deoxycorticosterone, 18 hydroxylation of corticosterone and dehydrogenation of 18 hydroxycorticosterone) were estimated after incubating transfected cells with ¹⁴C-deoxycorticosterone and analysing radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone and aldosterone. Both 11 and 18 hydroxylase activities were lower (19 and 12% respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats but no difference in systolic or diastolic blood pressures in either sex. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS but that, under normal environmental circumstances, we were unable to demostrate any influence of this mutation on blood pressure.