The ability of PSA1 embryonal carcinoma cells to differentiate when grown as clones on a monolayer of feeder cells was assessed using morphological criteria. The first appearance of a differentiated phenotype within a clone occurred at different times for individual clones after 10 days of culture, this being apparently unrelated to clone size or cell density. Those clones which showed no morphological evidence of differentiation after several weeks (about 5% of the clones observed) were selected and recloned with the aim of finding variant lines which were stably deficient in their differentiating ability. Undifferentiated clones - identified and selected after about 3 weeks of growth - were of three different types after recloning: those similar to control cultures of PSA1, those having delayed and reduced differentiation frequency, and those having variable frequencies of differentiation in replicate reclonings. The isolation of a variant with a more complete differentiation deficiency was accomplished by selecting ten nondifferentiating clones growing isolated in individual culture wells after 5 weeks of culture. One of these, T2H9, proved to be a stable, differentiation-deficient variant subline with less than 3% of its clones showing any morphological evidence of differentiation in five repeated reclonings. It was also determined that the frequency of undifferentiated clones in embryonal carcinoma cultures increased from 0.3% to 54% after 11 months of in vitro aging, i.e., approximately 200 cell doublings. The isolation of clonal embryonal carcinoma cell derivatives which are stable, heritable differentiation variants provides resources for somatic-cell genetic analysis of stem-cell pluripotency.