Uninucleate amoebae of Physarum polycephalum strain CL undergo apogamic development to form multinucleate plasmodia via an intermediate stage of large, uninucleate cells irreversibly committed to plasmodial development. This amoebal-plasmodial transition involves major changes in tubulin gene expression and the organization of microtubular structures. We analysed the expression of the betC locus, which encodes the plasmodial-specific beta 2-tubulin, during plasmodial development. A key question addressed was the timing of expression of betC in relation to the last open mitosis of the amoeba and the first closed mitosis of the plasmodium during the transition. Culture conditions were improved to yield partly synchronous differentiating cultures containing 50-60% committed cells, in order to facilitate biochemical analysis of development. Northern blotting indicated that betC RNA was virtually absent from amoebae and from early differentiating cultures. However, betC transcripts could already be detected in differentiating cultures containing only 0.1% of committed cells; the relative amount of betC transcripts increased as the percentage of committed cells in differentiating cultures increased. In fully developed plasmodia, there was at least a 330-fold increase in the betC transcript level compared to that in amoebae. We conclude that betC is activated during the amoebal-plasmodial transition immediately before or during the commitment event. Small amounts of beta 2-tubulin polypeptide could first be detected by Western blotting around the stage of the first closed mitosis. Thus beta 2-tubulin may participate in the first closed mitosis that committed cells undergo during their development into plasmodia.