Immunocytochemical analysis of small myogenic clones was used to compare the effects of fresh medium (FM) and conditioned medium (CM) on muscle differentiation. In order to compare the same population of cells, clones were initiated in FM and then switched to either new FM or to CM. Clones were fixed at 12-hour intervals up to 76 hours, then assayed for the presence of post-mitotic myoblasts by immunoperoxidase staining for muscle myosin heavy chain (MHC) or M-creatine kinase (MCK). In both media, myogenic cells occurred predominantly in homogeneous positive clones (all cells (MHC +/MCK +) which contained 2" cells. At 76 hours, the percentages of 1-, 2-, and 4-cell positive clones did not differ statistically in the two conditions; however, the percentages of 8- and 16-cell positive clones were significantly reduced in CM, and the percentages of small negative clones were concomitantly increased. We conclude from these data that CM affects myogenesis by slowing progression through a predetermined lineage rather than by changing the number of mitoses an individual cell will undergo before terminally differentiating. These results further support the idea that progress through the myogenic lineage is mediated by cell divisions.