Islets in most species respond to increased glucose with biphasic insulin secretion, marked by a sharp first-phase peak and a slowly rising second phase. Mouse islets in vitro, however, lack a robust second phase. To date, this observation has not been extended in vivo. We thus compared insulin secretion from conscious mice with isolated mouse islets in vitro. The arterial plasma insulin response to a hyperglycemic clamp was measured in conscious mice 1 wk after surgical implantation of carotid artery and jugular vein catheters. Mice were transfused using clamps with blood from a donor mouse to maintain blood volume, allowing frequent arterial sampling. When plasma glucose in vivo was raised from ∼5 to ∼13 mM, insulin rose to a first-phase peak of 403 ± 73% above basal secretion (n = 5), followed by a rising second phase of mean 289 ± 41%. In contrast, perifused mouse islets (∼75 islets/trial) responded with a similar first phase of 508 ± 94% (n = 4) but a smaller and virtually flat second phase of 169 ± 9% (n = 4, P < 0.05). Furthermore, the slope of the second-phase response differed significantly from zero in mice (2.63 ± 0.39%/min, P < 0.01), in contrast to perifused islets (0.18 ± 0.14%/min, P > 0.30). Mice also displayed pulsatile patterns in insulin concentration (period: 4.2 ± 0.4 min, n = 8). Conscious mice thus responded to increased glucose with biphasic and pulsatile insulin secretion, as in other species. The robust second phase observed in vivo suggests that the processes needed to generate second-phase insulin secretion may be abrogated by islet isolation.