Infection of mice with the protozoan parasite Leishmania major is an established model with which to study the in vivo development of CD4+ Th cell subsets. Interferon-gamma (IFN-gamma), produced by natural killer (NK) cells (AsGM1+, CD4-, CD8-, CD3-), regulates CD4+ T cell subset development and early resistance to L. major. Rapid Th1 cell development and resistance to infection occur in mice that develop an NK cell response early after infection (C3H and immunized BALB/c mice), whereas mice that lack an early NK cell response demonstrate delayed Th1 cell development and enhanced early disease (C57BL/6) or lack detectable Th1 cell development altogether and develop a progressive, fatal infection (BALB/c). Analysis of the requirements for NK cell activation in C3H mice revealed that the NK cell response is both interleukin-2 (IL-2) and IL-12 dependent. Although delayed IL-12 production in C57BL/6 mice precludes NK cell activation, the eventual development of a Th1 response appears to be IL-12 dependent. In contrast, concomitant production of inhibitory factors (IL-4, IL-10, and transforming growth factor beta) with IL-12 and IL-2 prevents NK cell activation in BALB/c mice. Together, these observations support a paradigm of in vivo Th1 cell development that involves IL-12-dependent stimulation of IFN-gamma production by NK cells.