To study the safety and efficacy of thymosin alpha1 in stimulating immune reconstitution in combination with highly active antiretroviral therapy (HAART), a phase II randomized, controlled open-label trial of subcutaneous thymosin alpha1 was undertaken for 12 weeks. Twenty clinically stable patients with viral loads <400 copies/ml and CD4 counts less than 200 cells/microl were randomized to receive 3.2 mg thymosin alpha 1 subcutaneous injections twice weekly or no injections for 12 weeks. CD4 and CD8 counts, CD45 RO+ and RA+ subsets and signal joint T cell receptor excision circles (sjTREC) in peripheral blood mononuclear cells (PBMCs) were measured every 2 weeks. Thirteen patients received thymosin alpha 1 and seven were controls. Thymosin alpha 1 was well tolerated and there were no serious adverse events. There was no significant difference between the thymosin alpha1 and control groups in CD4, CD8 and CD45 lymphocyte subset changes at week 12; however, PBMC sjTREC levels increased significantly in the thymosin alpha 1-treated patients compared to controls at week 12. In conclusion, the increase in PBMC sjTREC levels in patients taking thymosin alpha1 may represent enhanced immune reconstitution; however, the clinical benefits and long-term consequences remain to be determined.