Glucose and insulin are important regulators of islet beta-cell growth and function by activating signaling pathways resulting in transcriptional changes that lead to adaptive responses. Several immediate early genes have been shown to be rapidly induced by glucose-activated depolarization in islet beta-cells. The current studies address aspects of glucose-regulated transcription: 1) the number and characteristics of these genes, 2) if depolarization is the major mechanism, and 3) if glucose-stimulated insulin secretion is responsible, because insulin per se can activate transcription. Here, the expression profiles of glucose-responsive insulinoma cells 45 min after the addition of glucose, KCl to induce depolarization, or insulin were assessed by endocrine pancreas cDNA microarrays. Glucose activated more than 90 genes, representing diverse gene ontology functions, and most were not previously known to be glucose responsive. KCl activated 80% of these same glucose-regulated genes and, along with the effects of pretreatment with diazoxide, suggested that glucose signaling is mediated primarily via depolarization. There were >150 genes activated by insulin, and remarkably 71% were also regulated by glucose. Preincubation with a phosphatidylinositol (PI) 3-kinase inhibitor resulted in almost total inhibition of depolarization and insulin-activated transcriptional responses. Thus, through gene expression profiling, these data demonstrate that glucose and insulin rapidly activate a PI 3-kinase pathway, resulting in transcription of a common set of genes. This is consistent with glucose activation of gene transcription either directly or indirectly through a paracrine/autocrine effect via insulin release. These results illustrate that expression gene profiling can contribute to the elucidation of important beta-cell biological functions.