Using PNA and anti-Thy-1 fluorescent binding assays, T lymphocytes of the lizard, Chalcides ocellatus were phenotypically distinguishable into four subpopulations (PNA+ Thy-1-, PNA+ Thy-1+, PNA- Thy-1+ and PNA-Thy-1-), which seemed to be affected independently by endogenous steroid levels. Indeed, the size of PNA+ thymocytes is maximal and coincides with the low level of circulating cortisol during spring through summer and decreases gradually with the elevation of the cortisol level. On the other hand, as the endogenous testosterone (TS) level begins its physiological rise, lympholysis of Thy-1+ thymic cells begins in spring with gradual increase in size and with the decrease in TS levels. Among splenocytes and bone marrow lymphocytes, seasonal-dependent alterations in the size of both lymphocyte subpopulations seemed to correlate in part with the status of the thymus. Direct support of this observation was derived from subsequent in vitro studies with exogenous hydrocortisone (HC) and testosterone propionate (TP) treatments in spring and autumn. In all incidents, the data were indicative of the selective susceptibility of the PNA+ Thy-1- subpopulation to HC in the thymus and not in the periphery, and the susceptibility of the PNA- Thy-1+ subpopulation to TP in all three lymphoid organs tested. In vivo studies with a purified fraction of thymosin alpha 1 (T alpha 1) suggested that the PNA+ Thy-1- subpopulation in the different organs was the selective target for the action of T alpha 1. Finally, the dual treatment with T alpha 1 in vivo followed by TP or HC in vitro confirmed that TP-sensitivity was confined to the PNA- Thy-1+ and HC to PNA(+)-Thy-1- subpopulations in any of the three lymphoid organs.