The differential effects of in vitro cyclophosphamide (CY) on subpopulations of normal human peripheral blood lymphocytes involved in the pokeweed mitogen-induced plaque-forming cell (PFC) response against sheep red blood cells were examined. It was found that the plaque-forming B cells in this system are sensitive to CY over a wide concentration range including concentrations which have a minimal effect on overall cell viability. Kinetic experiments revealed that CY exerts its inhibitory effect on the PFC response only if added very early in culture. Thus, it appears that in vitro CY must exert its inhibitory influence on an early phase of polyclonal B-cell activation. When T-cell enriched (TCE) populations were incubated overnight with high concentration CY and then added back in co-culture to fresh autologous B cells, significant enhancement of PFC responses was observed suggesting a selective inhibition or elimination of a regulatory suppressor cell population found in TCE lymphocyte preparations. Helper T cells are relatively resistant to the inhibitory actions of CY. Thus, human B cells appear to be most sensitive to CY, followed in sensitivity by the suppressor cell populations in the T-cell fraction with relative resistance of the helper T cells. These observations have direct relevance in understanding the mechanisms of selective action of CY on normal human lymphocyte subpopulations with possible application to disease states in man.