We have studied the sera from 304 patients with insulin-dependent diabetes mellitus (IDDM) for steroid 21-hydroxylase (P450c21) autoantibodies by an in vitro translation and immunoprecipitation assay. Autoantibodies to P450c21 were found in 7 patients with IDDM (2.3%). When the IDDM patients with P450c21 antibodies were analyzed for their HLA, 6 of them (86%) belonged to the HLA DQB1*0201-positive group. A strong correlation (r = 0.91, P < 0.001) of immunoprecipitation results was observed with adrenocortical autoantibodies detected by indirect immunofluorescence, indicating that the adrenal autoantibodies in patients with IDDM were anti-P450c21 autoantibodies. The levels of P450c21 autoantibodies were significantly higher (P < 0.05) in patients with IDDM than in 13 (9 P450c21 autoantibody positive) patients with Addison's disease. Although the occurrence of P450c21 antibodies in IDDM was relatively low, antibody-positive patients had high antibody levels and may have an ongoing subclinical process of adrenal autoimmunity.