Pineal cells of the 8-day embryonic quail are multipotent cells which differentiate in vitro into skeletal muscle fibers, pigmented epithelial cells (PECs), lens cells and neurons. However, it was not yet clear whether precursor cells which gave such a wide repertoire of differentiation were single type or not. The present culture studies revealed that pineal cells were exclusively directed to ocular differentiation pathways by dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), suggesting a single type of precursor cell in the pineal body. DMSO directed pineal cells to differentiate into PECs. Co-administration of basic fibroblast growth factor (bFGF) with DMSO partially inhibited PEC differentiation and promoted lens cell differentiation. Northern blot analysis using cDNAs specific to PEC and lens cell confirmed this morphological observation. HMBA completely inhibited pigmentation of cultured pineal cells and markedly promoted lens cell differentiation. Ocular differentiation of pineal cells was accompanied with the loss of myogenicity. We discuss three possible pathways of lens cell differentiation from pineal cells. The agents which affect pineal cell differentiation seemed to modulate the cell-substrate interaction. And the interaction was suggested to be one of the environmental cues in the differentiation.