The signal requirements for proliferation of CD1+CD3- immature thymocytes have been studied in order to define whether this immature cell population could function despite the lack of the CD3/T-cell receptor complex. We found that CD1+CD3- cells proliferate upon stimulation with anti-CD28 monoclonal antibody as well as with a pair of anti-CD2 monoclonal antibodies in the presence of low doses (0.5 ng/ml) of phorbol-13-myristate-12-acetate and/or recombinant interleukin-2. A minor fraction of CD3+ cells (15%-20%) was also present in the proliferating cell population originating from CD1+CD3- thymocytes stimulated with phorbol-13-myristate-12-acetate and recombinant interleukin-2, either in the presence or in the absence of specific monoclonal antibodies. We further observed that the anti-CD3 monoclonal antibody did not induce the proliferation of CD1+CD3- cells, as expected, and efficiently triggered unfractionated or CD1+CD3+ thymocytes only if exogenous recombinant interleukin-2 was provided. Unexpectedly, we noted that highly purified (greater than 99%), CD1+CD3- immature thymocytes could mobilize calcium via CD3, besides CD2 and CD28 surface molecules, suggesting that a minor undetectable fraction (less than 1%) of CD3+ cells was still present in the purified CD3- population. Nevertheless, the preferential expansion of CD3-CD8+ cells (about one-third of proliferating cells) after triggering via CD28, and to a lesser extent via CD2, support the notion that the alternative pathways of T-cell activation are actually functional in CD1+CD3- immature thymocytes.