We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J. Neurochem. 68, 882-885). We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [(14)C]hexanoyl ceramide to [(14)C]hexanoyl GlcCer is detected, but there are no changes in the rate of [(14)C]hexanoyl sphingomyelin synthesis. In vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [(14)C]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [(14)C]hexanoyl GlcCer synthesis. The increased rate of [(14)C]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.