3'-Azido-2',3'-dideoxythymidine (AZT) is extensively used in the treatment of human immunodeficiency virus (HIV) infection. Currently, it is debated whether AZT should be offered to symptomless HIV-infected individuals in the hope of delaying or even preventing progression to acquired immunodeficiency syndrome (AIDS). However, before the chronic use of AZT, it is essential to establish whether this drug alters the differentiation and functions of T cells particularly because HIV infects CD4+ T cells, as well as investigate whether AZT would alter the T cell response to HIV-encoded antigens. In the current study, therefore, we investigated the effect of administration of AZT into mice for 7-14 days on T cell differentiation in the thymus and the ability of T cells to respond to HIV antigens in the periphery. Our data demonstrated that AZT, when administered orally at 500 mg/kg body wt or higher concentrations for 14 days, caused marked thymic involution with significant decrease in the percentage of CD4+CD8+ T cells and increase in the percentage of CD4-CD8-, CD4+, and CD8+ T cells. AZT treatment did not affect the cellularity of the spleen or the ratios of T cells in the periphery. Also, splenic T cells from AZT-treated mice did not demonstrate marked decrease in their ability to respond to mitogens in vitro. However, when AZT-treated mice were immunized with foreign antigens, such as gp120 of HIV or conalbumin, the T cells demonstrated significant decrease in their ability to respond to these antigens. When AZT was added to cultures, it was found to inhibit the proliferation of T cells to mitogens as well as the differentiation of cytotoxic T lymphocytes (CTL). Interestingly, addition of exogenous IL-2 to CTL cultures reconstituted the decreased CTL response. Furthermore, administration of IL-2 into AZT-treated mice could also reconstitute the decrease in thymic cellularity induced by AZT. These data indicate that AZT, when present at the time of T cell differentiation or responsiveness to antigens in vivo, can mediate significant inhibition of such functions thereby suggesting that AZT may affect the immune response to HIV antigens.