Week-old rat cerebellar granule cells were grown in the presence of the cell-permeable calcium chelating agent BAPTA-acetoxy methyl ester (BAPTA-AM) for the first 8 h in vitro. There was a dose-dependent inhibition of process outgrowth with an IC50 of approximately 5 microM. Neurite outgrowth could be partially recovered by the addition of N-methyl-D-aspartate (NMDA; 50 microM) to BAPTA-AM-treated cells. Phorbol ester stimulation of treated cells evoked a profound inhibition of neuritogenesis compared to a stimulatory effect on control cultures. The inhibition of growth caused by phorbol esters could not be reversed by NMDA co-addition. Neurites extended by BAPTA-AM-treated granule cells were thinner than in control cultures and did not form elaborate growth cones even when growth was stimulated by NMDA. The distribution of tyrosinated and acetylated alpha-tubulin in the processes of BAPTA-AM-treated cells appeared similar to that in controls. However, rhodamine-phalloidin labelling of microfilaments in the cell cultures emphasised the loss of an elaborate actin-rich growth cone in BAPTA-AM-treated cells even when neurite formation was partially recovered. These results indicate the importance of [Ca2+]i in the production of neurites from cerebellar granule cells in vitro.