A water soluble benzodiazepine, clorazepate, has been used to establish the point of benzodiazepine proliferative arrest in the rat C6 glioma. Clorazepate inhibited C6 proliferation in a dose-dependent manner with an IC50 value of 280 microM, as judged by a nuclei counting procedure. Release of cells from a 48 h exposure to 350 microM clorazepate, at which over 70% of the cells were arrested, resulted in a synchronous entry into S phase 8-9 h later, as evidenced by a sharp increase in the incorporation of [3H]thymidine. This restriction point was demonstrated to be 2-3 h into the G1 phase by measuring the length of G1 in synchronized populations of C6 cells obtained by selection of mitotic figures from an asynchronous culture. Synchronous arrest of C6 by clorazepate required an exposure period of 24-36 h, approximately twice the doubling time of the cell line. A morphological study confirmed an early G1 point of proliferative arrest. Clorazepate synchronized cells exhibited a uniform morphology with the majority of cells assuming a configuration representative of anchorage-dependent cells in an early phase of attachment. The majority of cells were somewhat rounded and attached to the substratum by cytoplasmic 'skirts' with punctate structures which may represent focal adhesion points.