The HIT cell is a variably glucose-responsive clonal line of pancreatic islet β-cells. To ascertain whether insulin responsiveness to glucose, arginine, isoproterenol, forskolin, and K+ varied in a predictable fashion, full concentration-response curves with these agonists were examined with cells from a span of 25 passages. Basal and stimulated cAMP metabolism were also examined. The findings indicate that insulin responses to glucose diminish progressively with increasing passage number and that studies of glucose-induced insulin secretion should be limited to passages 81 and earlier. This defect in insulin secretion is a general rather than a glucose-specific phenomenon in that insulin responses to the other nonglucose secretagogues also diminished with increasing passage number. All changes in glucose-stimulated responses were limited to diminutions in maximal responses; no alterations in apparent half-maximal effective concentrations (EC50s) were observed. In contrast to the continually diminishing insulin responsiveness observed, hormone inhibition by somatostatin of insulin secretion and basal cAMP metabolism remained intact throughout the passages examined. Interestingly, a reciprocal and cAMP responsiveness was observed. Dramatic cAMP responses to isoproterenol and forskolin were observed with the later passages. We conclude that loss of insulin responsivity in HIT cells is a passage-dependent process that is serial rather than sporadic and global rather than glucose specific. Dramatic reciprocal changes in cAMP metabolism occur in the later passages.