In vivo β-cell function tests are used increasingly in humans during the preclinical phase of insulin-dependent diabetes mellitus (IDDM), but the severity of the β-cell loss responsible for the abnormalities seen in these tests is unknown. We have measured several physiological β-cell function tests—fasting plasma glucose, glucose disappearance constant, fasting insulin, acute insulin responses to arginine (AIR_arginine) and glucose (AIR_glucose), and glucose potentiation of AIR_arginine (∆ AIR_arginine/∆G) and two direct objective measurements (pancreatic insulin content [PIC] and quantitative β-cell mass)—in adolescent male baboons (Papio anubis/cyanocephalus). We have correlated in vivo measurements obtained within 3 days after the animals were killed with in vitro estimates of PIC and β-cell mass in 15 animals, (2 nondiabetic requiring insulin treatment and 13 after varying doses of streptozocin to induce degrees of β-cell damage ranging from normoglycemia to severe hyperglycemia). There was a strong linear correlation between β-cell mass and PIC (r = 0.79, P < 0.001). Physiological measures of β-cell function were significantly correlated with both PIC and β-cell mass. The correlations between physiological measures and β-cell mass were linear and intercepted the β-cell mass axis at 0.15–0.2 g, suggesting that in vivo measures of β-cell function approach 0 when there is still ∼40–50% of the β-cell mass detectable histologically. With PIC, the linear correlations intercepted the axes close to 0. These findings provide considerable validity to the measurements of β-cell function used in preclinical IDDM in humans. Our data suggest that such physiological measurements give an accurate valid reflection of changes in β-cell mass and pancreatic insulin content.