Rat peritoneal mast cells (MC) cocultured with 3T3 fibroblasts (MC/3T3) were activated by compound 48/80 to release most of their histamine content (approximately 90%). These activated MC could be kept viable in vitro for as long as 2 weeks, during which their regeneration had been followed. As early as 3-4 hr following activation, the MC started reacquiring their resting morphology. In 1-2 weeks they were more densely granulated grossly resembling nonactivated, control MC. Reactivation of MC/3T3 with compound 48/80 on Day 7 or 14 of culture resulted in a percentage histamine release which was similar to that of Day 0, and to control MC/3T3, indicating that these activated MC were functionally responsive. Activated MC/3T3 could partially resynthesize histamine during the 14 days of culture following activation. On Day 7 their histamine content was approximately 23% and on Day 14 approximately 40% of the initial amount they had on Day 0, prior to activation. Thus, MC/3T3 activated by compound 48/80 in vitro can regenerate by resuming their initial morphology, becoming responsive to a subsequent reactivation and resynthesizing histamine.