This study examined the metabolism of arachidonic acid (AA) by cytochrome P450 (CYP) enzymes in isolated glomeruli and the effects of selective inhibitors of the synthesis of 20-HETE and EETs on glomerular permeability to albumin (P_alb). Glomeruli avidly produced 20-HETE, EETs, DiHETEs, and HETEs when incubated with exogenous AA. N-hydroxy-N`-(4-butyl-2methylphenyl) formamidine (HET0016, 10 µM) selectively inhibited the formation of 20-HETE by 95% and increased P_alb from 0.00 ± 0.08 to 0.73 ± 0.10 (n=43 glomeruli, 4 rats). Addition of a 20-HETE mimetic, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid (20-5, 14-HEDE, 1 µM), opposed the effects of HET0016 (10 µM) to increase P_alb (0.21 ± 0.10, n=36 glomeruli, 4 rats). Preincubation of glomeruli with exogenous AA to increase basal production of 20-HETE had a similar effect. We also examined the effect of an epoxygenase inhibitor, N-methylsulphonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH, 5 µM), on P_alb. MSPPOH (5 µM) significantly increased P_alb but had no effect on the synthesis of EETs in glomeruli incubated with AA. However, MSPPOH (5 µM) selectively reduced epoxygenase activity by 50% in glomeruli incubated without added AA. Pretreatment with 8, 9-EET (100 nM) attenuated the effects of MSPPOH (5 µM) on P_alb. These results indicate that glomeruli produce 20-HETE, EETs, DiHETEs and HETEs and that endogenously formed 20-HETE and EETs play an essential role in the maintenance of the glomerular permeability barrier to albumin.