Isolated human islets were examined for the rates of conversion and release of newly formed (pro)insulin-like peptides. The rate of proinsulin (PI) conversion was 2-fold slower in human β-cells (t_1/2 = 50 min) than in rat β-cells (t_1/2 = 25 min). During the first hour following labeling of newly synthesized proteins, PI represented the main newly formed hormonal peptide in the medium; its release was stimulated 2-fold over the basal level by 20 mmol/L glucose. During the second hour, newly synthesized hormone was mainly released as insulin, with 10- to 20-fold higher rates at 20 mmol/L glucose. Prolonged preculture of the islets at 20 mmol/L glucose did not delay PI conversion, but markedly increased the release of newly formed PI, des^31,32-PI, and insulin at both low and high glucose levels. Our data demonstrate that 1) the release of PI provides an extracellular index for the hormone biosynthetic activity of human β-cells; 2) an acute rise in glucose exerts a stronger amplification of the release of converted hormone than in that of nonconverted hormone; and 3) prolonged exposure to high glucose levels results in an elevated basal release of converted and nonconverted PI; this elevation is not associated with a delay in PI conversion, but is attributed to the hyperactivated state of the human β-cell population, which was recently found to be responsible for an elevation in basal rates of hormone synthesis. These in vitro observations on human β-cells provide a possible explanation for the altered circulating (pro)insulin levels measured in nondiabetic and noninsulin-dependent diabetic subjects.