Cytochrome b561 is an electron transfer protein unique to neuroendocrine secretory vesicles. The Southern blot hybridization shows that it is a single copy gene highly conserved throughout phylogeny. The transcription unit spans approximately 11 kilobases, and heterologous transcription sites are located 404 bases 5' to the translation initiation codon. The sequence of the 5'-flanking region is GC-rich and lacks a typical TATA box at the usual position. However, it has a CAAT sequence at -132 and potential recognition sequences for several transcription factors including SP1, GR-PR-MMTV, AP4, gERE, JCV repeat, AP2, and NF-kappa B. Each of the five transmembrane segments are encoded by five consecutive exons. This corroborates the five-transmembrane model proposed for human, mouse, and Xenopus rather than six proposed for bovine. The cytochrome was found to be highly expressed in colon cancer cell lines, T cell lymphomas, and K-562 cell lines. However, in B-cell lymphomas such as Burkitt's and Daudi, the cytochrome b561 expression was completely shut down. The results in this report are the first to demonstrate the structural organization and regulatory sequences of the cytochrome b561 gene encoding an integral membrane protein of neuroendocrine storage vesicles of neurotransmitters and peptide hormones. Unexpected results on cytochrome b561 expression in cells of lymphocytic origin and its complex regulation in tumor cells provide new insights into cytochrome b561 gene regulation.