Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing 10–60 μg 14,15-epoxyeicosatrienoic acid/ml (14,15-EET) had no detectable effect on blood flow. However, 25 ±3 μg 11,12-EET/ml and 36 ±2 μg 8,9-EET/ml caused increases (134 ± 8% and 127 ± 6%) that were similar to those elicited by 8 ± 2 μg adenosine/ml (138 ± 12%). Furthermore, the increases (275 ± 38%) produced by 32 ± 6 μg 5,6-EET/ml exceeded those elicited (160 ± 10%) by a similar concentration (27 ± 3 μg/ml) of adenosine. Thus, a structure-activity relationship is suggested. Nevertheless, these values probably underestimate the potency of the EETs because the vasoactivity was reduced by contact with water. The activity of the cyclooxy-genase pathway seemed to limit the formation of vasoactive quantities of EETs, or other nonprosta-noids, from exogenous arachidonate in the serosa but not the mucosa. A bolus (1.3 ± 0.2 mg/ml) or continuous application (122 ± 45 μg/ml) of arachidonate caused blood flow increases (236 ± 14% or 229 ± 27%) that were almost eliminated (129 ± 5% or 121 ± 9%) by a cyclooxygenase inhibitor; the residual response was abolished by a cytochrome P450 inhibitor. However, cytochrome P450 inhibitors alone did not attenuate the arachidonate response. In contrast, a continuous application of 194 fig arachidonate/ml to the mucosa caused a markedly smaller blood flow increase (119 ± 8%) and cyclooxygenase inhibitors potentiated (132 ± 8%), rather than reduced, this response. We conclude that EETs are a labile class of vasodilators with a potency comparable to adenosine in the intestinal microcirculation. Indirect evidence suggests regional differences in the formation of vasoactive quantities of arachidonate metabolites within the intestinal wall.