There is an unexplained, marked regional heterogeneity in perfusion within single skeletal muscles both in dogs and rabbits. We asked if a similar distribution of perfusion was present within cat muscles. If present, we wanted to assess the possible roles of nitric oxide (NO) mediated vasodilation and citrate synthase (CS) activity for the regulation of this perfusion pattern. Perfusion was determined in 0.25 g regions within the gastrocnemius muscles by trapping of microspheres. We studied awake or anesthetized cats before and during inhibition of NO-formation using N-monomethyl-L-arginine. The CS activity was determined in homogenates of these regions. The coefficient of variation corrected for the Poisson distribution of microspheres (CVc) for the regional perfusion averaged 0.39. Despite a 25% reduction in perfusion to the whole muscles as compared to control, the uneven distribution of perfusion was not affected by blocking NO formation. Regional perfusion was not correlated to regional CS activity. Even if the regional distribution of CS activity also showed a scatter, mean coefficient of variation corrected for methodological error = 0.20, it was markedly less than that for perfusion. We conclude that neither NO vasodilation nor CS activity play an important role in the regulation of the regional perfusion pattern within single cat muscles.