Cells were isolated from the outer medulla of the rabbit kidney, primarily from the thick ascending limb of Henle's loop (mTALH). These mTALH cells are heavily invested with a cytochrome P450-linked monooxygenase that represents the third pathway by which arachidonic acid is metabolized. After cell separation, approximately 80% of the cells proved to be mTALH in origin, based on electron microscopic criteria and immunofluorescent localization of Tamm-Horsfall protein, a specific marker for mTALH cells. The specific activity of alkaline phosphatase, a marker for proximal tubular cells, decreased threefold after separation of mTALH cells from outer medullary cells, associated with a fourfold increase in the capacity of the separated mTALH cells to metabolize arachidonic acid. Incubation of mTALH cells with 14C-arachidonic acid resulted in formation of oxygenated metabolites, identified as two peaks (P1 and P2), which accounted for 30 to 40% of the recovered radioactivity. Formation of prostaglandin E2 and F2 alpha accounted for only 3 to 5%. The chromatographic retention times of P1 and P2 were different from products of lipoxygenases. An inhibitor of cytochrome P450-dependent enzymes, SKF-525A (50 microM), reduced product formation by mTALH cells by more than 70%, while induction of cytochrome P450 increased product formation. Formation of P1 and P2 by cell-free homogenates of mTALH was totally dependent on the presence of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), which suggests a NADPH-dependent cytochrome P450-linked monooxygenase pathway. Vasopressin and calcitonin (10(-10) M to 10(-7) M) stimulated release of arachidonic acid metabolites from mTALH cells.(ABSTRACT TRUNCATED AT 250 WORDS)