Correlations of peripheral blood thymus-dependent lymphocyte (T cell) levels as determined by the formation of E rosettes with sheep red blood cells and cell-mediated immunity emphasize the need for an objective means of determining T cell levels. A technique for determining T cell levels by cytofluorographic analysis utilizing glutaraldehyde fixation of the cells was devised and demonstrated to correlate with T cell levels as determined by light microscope counting. The in vitro assay of lymphocyte reactivity to PHA and the assay for T cell levels derived in this laboratory were shown to correlate with tumor burden in patients with melanoma: patients with localized or regional disease did not differ from normals; however, those with disseminated disease had mean levels lower than normals. Healthy subjects with a history of cigarette consumption of 20 pack-years or less or those who were less than 40 years of age had increased in vitro lymphocyte reactivity and T cell levels compared to nonsmokers. After cessation of smoking, lymphocyte reactivity returned to normal within 3 months; however, T cell levels remained elevated compared to normal controls. These observations define another important variable that must be considered in the interpretation of these in vitro correlates of cell-mediated immunity.