Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.