TCR gamma delta + cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their maximum proliferation. MNC were cultured for 6 days with Streptococcus pyogenes, and for 3 days with T cell mitogens, PHA and anti-CD3 mAb, in medium supplemented with various concentrations (0.05-50%) of human sera. TCR gamma delta + and TCR gamma delta- CD2+3- double negative cells induced by Str. pyogenes required high concentrations of sera (greater than 6%) for their proliferation. Moreover, increased sera (up to 50%) greatly augmented their proliferation. On the other hand, TCR alpha beta + cell proliferation induced by T cell mitogens was supported by a small concentration (even 0.1%) of the sera, and the addition of high concentrations of sera (greater than 6%) somewhat suppressed responses. Similar serum requirement patterns were evident for the induction of cytotoxic cells. These results clearly demonstrated the existence of an appropriate culture condition for the proliferation of TCR gamma delta + cells induced in vitro.