We have established an assay based on gas chromatography-mass spectrometry to profile and quantitate endogenous cytochrome P450 monooxygenase (P450)-hydroxyeicosatetraenoic acids (HETEs) exiting the isolated perfused rabbit kidney in response to hormonal stimulation. In response to angiotensin II (Ang II) P450-derived HETEs (16-, 17-, 18-, 19- and 20-) are released from the isolated Kreb's perfused rabbit kidney. Ang II produced a several-fold increase in the levels of P450-HETEs above basal levels in both urinary (such as for 20-HETE from 0.93 +/- 0.7 to 2.31 +/- 0.9 ng/min) and venous (from 0.1 +/- 0.05 to 0.3 +/- 0.05 ng/min) effluents. However, inhibition of P450, which reduced basal release, did not prevent Ang II-induced release of P450-AA products from the rabbit kidney; for example, urinary 20-HETE in the presence of 17-ODYA (1 microM) was undetectable and increased to 0.93 +/- 0.4 ng/min with Ang II and venous 20-HETE increased from 0.06 +/- 0.03 to 0.24 +/- 0.07 ng/min. Similar results were obtained with clotrimazole (1 microM). As 16-, 18-, 19- and 20-HETEs are vasodilators in the rabbit kidney and 16- and 17-HETEs inhibit proximal tubular ATPase activity, we investigated their possible sites of esterification. Cortical and medullary lipids were extracted, separated by HPLC and P450-HETEs quantitated following alkaline hydrolysis. The P450-HETEs were incorporated into both neutral lipids (NL) and phospholipids [phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine (PC)]. However, the assignment of a HETE to a specific phospholipid pool must be regarded as tentative as the appropriate standards containing P450-HETEs in the Sn-2 position (such as 20-HETE-PF., 20-HETE-PC, etc.) were not available. Esterified HETEs were found in larger quantities in the cortex as compared to the medulla (34.40 +/- 1.12 versus 22.76 +/- 0.53 ng/g). The PI fraction in the cortex yielded the largest quantity of HETEs and the PC fraction the lowest. In the medulla, the largest quantities of esterified HETEs were found in neutral lipids and only slightly lesser amounts in PE and PI. Esterified 18-HETE was localized only to the NI fraction. This fraction also contained the other HETEs, 19- and 20-HETE being the most abundant. Notably only 16- and 17-HETE were present in PF, whereas, 19- and 20-HETE were also present in PI, PS and PC. Thus, P450-HETEs, like EETs are stored in the kidney and are, presumably, subject to release by peptide activation of acylhydrolases.