Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).