Impairments in the respiratory burst and stimulus-response coupling were studied with respect to the increased rate of cell replication that occurred in HL60 cells during repetitive passages in cell culture. During a 45-week period of culture, HL60 cells developed a progressive increase in rate of replication. Concomitantly, undifferentiated cells developed an impairment in ATP-induced calcium mobilization. The percentage of cells that could be differentiated with dimethyl sulfoxide progressively diminished. Differentiated cells developed an impairment in both the respiratory burst and secretion of beta-glucuronidase. In addition, regulation of the respiratory burst by cAMP agonists including isoproterenol, adenosine, and prostaglandin E2 was reduced in rapidly proliferating cells. Thus, multiple changes in stimulus-response coupling occur during cell culture in association with an increase in rate of cell replication. It may be important to recognize progressive impairments in cell function in studies using repetitive samples of HL60 cells from a continuously maintained cell population. The observed impairments in stimulus-response coupling may be relevant to unregulated cell growth in neoplastic disease.