Human T micron, T gamma, and T PHI lymphocyte subpopulations have the capacity to respond to phytohemagglutinin (PHA) in vitro with proliferation and the production of a pH 2 and heat-labile gamma interferon. This occurs both when the subsets are isolated by direct rosetting techniques or by negative selection. Macrophages enhance the production of the gamma interferon by each lymphocyte subset and do not themselves produce gamma interferon in response to products of PHA-activated lymphocyte subsets. Thus our studies indicate that subpopulations of T lymphocytes known to differ with regard to morphology, surface receptors, RNA content, response to corticosteroids and X-irradiation, and other functional capabilities do not differ with regard to their capacity to produce gamma interferon.