Limiting-dilution culture of murine Ly-2+ T cells with concanavalin A (Con A) and irradiated spleen filler cells produces, with high efficiency, cytolytic T lymphocyte (CTL) clones. With most mouse strains (including CBA and C57BL/6) the specificity of these CTL clones drops after day 6 of culture, so that by day 9 the majority of clones can lyse most murine target cells, whether syngeneic or allogeneic. The rate of specificity degradation and relative target cell preference varies with the mouse strain. Some strains (e.g. BALB/c) do not show this effect and CTL clones remain specific to day 9. Many low natural killer (NK) cell strains (e.g. C57BL/6J.bg) maintain CTL specificity in such cultures, but the correlation between CTL specificity and NK status is not absolute. Growth of BALB/c precursor cells on CBA filler cells leads to specificity degradation in the BALB/c CTL clones; however, the specificity of CBA-derived CTL clones is not maintained by growth on BALB/c fillers. The results suggest that specificity degradation is induced in the developing CTL-clone by factors in the culture environment, perhaps a soluble lymphokine (a differentiation factor) or by an infectious agent (an endogenous mouse virus). Although such CTL specificity loss may render many limiting dilution studies of the CTL specificity repertoire invalid, the problem may be bypassed by an appropriate choice of mouse strain.