Cytochrome b561 family was characterized by the presence of "b561 core domain" that forms a transmembrane four helix bundle containing four totally conserved His, residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL Won b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D(fungi), E (animals/TSF), F (plants+DoH), and G (SDR2). In groups A, both motif 1, {FN(X)HP(X)(2)M(X)(2)G(X)(5)G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, (LFSWHP(X)(2)M(X)(3)F(X)(3)m(X)EAIL(X)SP(X)(2)SSI, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X)(3)Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X)(2)G(R/Y)} were found in group F at different regions from those of motifs 1 and 2. The "DoH" domain common to the NH2-terminal region of dopamine beta-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561. (c) 2005 Elsevier B.V. All rights reserved.