Earlier studies have shown that 17 alpha-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc) are the main autoantigens recognized by sera from patients with Addison's disease associated with autoimmune polyglandular syndrome type I (APS I). In this study we tried to identify the autoantigenic epitopes on P450c17 and compared the identified sequences with corresponding regions in two other adrenal autoantigens, P450scc and P450c21. A series of P450c17 cDNA fragments was expressed in Escherichia coli and the recognition of the corresponding protein fragments by patient sera was tested by immunoblotting. Four distinct epitope regions (ER) were found: ER1 (amino acids 122-148), ER2 (280-304), ER3 (396-432) and ER4 (466-508). B cell epitope prediction analysis showed that the four identified ERs were all located in regions of high predicted antigenicity. Homology search revealed that ER3 and ER4 but not ER1 and ER2 were related to similar sequences on P450c21. No significant homologies with P450scc in these regions were found. Although all three P450 cytochromes are genuine autoantigens this finding suggests that the autoantibody reaction against P450c17 and P450c21 can partly be a result of immunological cross-reactivity.