We studied the effects of nerve growth factor (NGF), fibroblast growth factor (FGF), and dibutyryl-cAMP (dbcAMP) on rat pancreatic β-cell morphology and of NGF and dbcAMP on insulin secretion. After 2 wk in culture, nearly 3% of β-cells extended neurite-like processes spontaneously; when cells were treated with NGF, almost 30% of them extended processes. In the presence of dbcAMP, almost all β-cells flattened, and the extension of neurite-like processes was more pronounced in fetal than in adult cells. The most prominent effect, regardless of age, was observed in cells treated with NGF and dbcAMP together, since the percentage of neurite-like bearing β-cells increased to 50%. β-cells cultured under these conditions maintained their immunoreactivity to insulin and nearly all β-cells and their neurite-like processes were also positive to GABA, tubulin, tau protein, and N-CAM. FGF increased the percentage of adult β-cells bearing neurite-like processes to 13%, and FGF and dbcAMP applied together to 40%. β-cells treated with NGF and dbcAMP for 5 to 7 d preserved their capability to secrete the hormone in response to different extracellular glucose concentrations. Insulin secretion of dbcAMP-treated β-cells was 2.5-fold higher than in control cells. NGF-treated cells were able to discriminate between different glucose concentrations, a property lost in control cells with time in culture.