Effects of polyamine depletion by alpha-difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles relevant for protein synthesis was observed upon culturing in the presence of 4,5,6,7-tetrahydro-isoxazole[5,4-c]pyridin-3-ol (THIP) for 4 days. The intracellular concentrations of putrescine, spermidine, and spermine in these cultures were similar to the concentrations of the polyamines observed in cultures grown in a plain culture medium for 1, 2, 3 or 4 days, respectively. Upon culturing in the simultaneous presence of THIP and DFMO, the concentrations of putrescine and spermadine were reduced to less than 20% of the levels in the controls. This depletion was associated with a severely impaired morphological development of the granule cell cultures. Thus, the number of neurite extending cells was reduced to 50% of the number in the control cultures upon culturing in the presence of DFMO alone or in combination with THIP. Moreover, the THIP mediated increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO.