The present experiments were designed to assess the extent to which the β-cell microtu-bular-microfilamentous system contributes to the release of insulin evoked by glucose or sulfonylurea in the isolated perfused rat pancreas. Deuterium oxide (100%, v/v), which is thought to interfere with micro-tubular-microfilamentous function, was found to suppress insulin release during both the early and late phases of the secretory process. The inhibitory effect of D2O was reversible. When the pancreases) were first exposed to cytochalasin B (10 µg/ml) and, thereafter, (to both cytochalasin B and D2O, an escape)phenomenon from the D2O-induced inhibition was) invariably observed in response to either glucose or sulfonylurea. This escape phenomenon supports the view that the inhibitory effect of D2O on insulin)release is mainly due to a functional sideration of the microtubular-microfilamentous system. The present data suggest, therefore, that such a system is actively, involved in both phases of insulin release. (Endocrinology95: 1518, 1974)