Rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) were activated with Ag or with anti-IgE antibodies in the presence of Ca2+, and their responsiveness to a second similar challenge was evaluated. MC/3T3 were presensitized with IgE anti-DNP antibodies and activated with DNP-human serum albumin. When these MC/3T3 were reactivated with the same Ag 2 and 6 h later, they released only a minimal percentage of histamine. A gradual recovery of responsiveness was detected during the first 7 days after activation, and a full recovery was attained by days 14 to 21. A similar pattern of unresponsiveness was observed when MC/3T3 were challenged and rechallenged with cercarial Ag after presensitization with anticercarial serum. Activation of MC/3T3 with one Ag (DNP-human serum albumin or cercarial) and rechallenge 3 days later with the other Ag did not overcome the state of partial unresponsiveness. Challenging MC/3T3 with anti-IgE led to a subsequent unresponsiveness to rechallenge with the same ligand, regardless of whether or not the cells were presensitized with IgE antibodies. Cross-linkage with anti-IgE resulted in a more intense and prolonged state of unresponsiveness in comparison with that observed with Ag. When MC/3T3 activated with anti-IgE were rechallenged with various IgE-independent agents they released a percentage of histamine comparable to that of control cultures challenged with these secretagogues for the first time. MC/3T3 partially resynthesized their histamine content during the two-week period after activation. Our results suggest that MC undergo a temporary state of "physiologic" unresponsiveness after immunologic activation in the presence of calcium ions.