To establish whether the heterogeneous secretion of glucose-stimulated beta-cells correlates with a different biosynthetic activity, we have studied the secretion and biosynthesis of the very same beta-cells by combining a hemolytic plaque assay with autoradiography. After a 10-min incubation in 2.8 mM glucose, 52 +/- 2% of dispersed rat beta-cells incorporated [3H] leucine into newly synthesized proteins, as revealed by autoradiographic labeling. When the incubation was performed in 16.7 mM glucose, larger (P less than 0.02) proportions (92 +/- 4%) of plaque-forming, i.e. insulin-secreting, and nonplaque-forming beta-cells (74 +/- 4%) were autoradiographically labeled. Labeled and unlabeled beta-cells were stimulated to secrete insulin during a 30-min incubation in 16.7 mM glucose, as revealed by the larger (P less than 0.001) formation of hemolytic plaques. Under these conditions, autoradiographically labeled beta-cells were recruited preferentially (P less than 0.01) and secreted more (P less than 0.04) than unlabeled beta-cells. Analogous observations were made with beta-cell pairs. Under glucose stimulation, pairs comprising two autoradiographically labeled beta-cells secreted more (P less than 0.004) than pairs comprising one or no labeled beta-cells. The data indicate that under glucose stimulation, 1) secreting and nonsecreting beta-cells increase protein biosynthesis; 2) biosynthetically active and inactive beta-cells increase insulin secretion; 3) beta-cells synthesizing new proteins release insulin preferentially; and 4) contact decreases the biosynthetic and secretory heterogeneity of beta-cells.