In the present study we have used a reverse hemolytic plaque assay and frequency distribution analysis to characterize the insulin secretory response of individual rat pancreatic B-cells in culture. Heterogeneous secretory behavior was observed with a multimodal distribution, suggesting the existence of B-cell subpopulations. Insulin secretion in response to increased extracellular glucose involved both amplification of secretion by individual cells as well as recruitment of previously silent B-cells.