Cloning of a human T cell leukemic cell line, HSB.2, was performed by a limiting dilution method to obtain clones with high levels of IL 2 production. None of the subclones that were obtained produced IL 2 constitutively, and only a low level of IL 2 was produced by the stimulation of these subclones with phytohemagglutinin (PHA) alone. High levels of IL 2 production (greater than 300 U/ml) were observed in several clones when stimulated with a cocktail of PHA and IL 1. Among them, HSB.2-A7-D2, A7-D9, or C5-B2 subclones, which were selected after cloning twice, were most effective in IL 1-dependent IL 2 production. HSB.2 subclones exhibited IL 1-dependent production of a variety of lymphokines other than IL 2, e.g., interferon-gamma (IFN-gamma), B cell growth factor (BCGF), and colony-stimulating factor (CSF). We observed that subclones with high IL 2-producing capacity tended to produce high levels of IFN-gamma or BCGF as well, while the capacity of CSF production was not parallel to these properties. Although several subclones were found to produce IFN-gamma and BCGF simultaneously with minimal IL 2 activity, no subclones with an exclusive BCGF production were obtained. Furthermore, when supernatants from the stimulated A7-D9 subclone were applied to an Ultro-gel AcA54 gel chromatography, it was revealed that IL 2 activity (m.w. 17K to 18K) and IFN-gamma (40K to 45K) were clearly separated, whereas two peaks of BCGF activity coincided with each peak of IL 2 and IFN-gamma, respectively. On the other hand, CSF activity was eluted at a different peak (30K to 35K). These data indicate that IL 2, IFN-gamma, and CSF activities are based on distinct molecules, whereas BCGF activities are indistinguishable from IL 2 and IFN-gamma. The HSB.2 subclones thus selected will provide a useful model for delineating the mechanism of IL 1-dependent lymphokine(s) production, and are a promising candidate for better lymphokine(s) producers.