We recently showed that thiamine uptake by neuroblastoma cells is mediated by two saturable transport system: the first with high affinity for thiamine (Km = 35 nM) is blocked by veratridine; the other, with low affinity is blocked by Ca2+. The driving force for thiamine uptake is its phosphorylation to thiamine diphosphate (TDP) by thiamine pyrophosphokinase and subsequent binding of this cofactor to apoenzymes. Our results suggest that cells of neuronal origin possess mechanisms regulating the intracellular concentration of thiamine. At low external thiamine, the vitamin is taken up by a high-affinity transporter and pyrophosphorylated in thiamine diphosphate (TDP): this is the TDP pool of slow turnover. An intraover extracellular concentration gradient of free thiamine is observed at low external concentration of the vitamin. At higher external thiamine concentration, TDP accumulation is limited by the binding capacity to the apoenzymes and unbound TDP (i.e. a small pool of fast turnover) is quickly hydrolyzed. Thiamine is slowly released by the cells by at least two different mechanisms. The first, accounting for a maximum of 50% of total thiamine release, is stimulated by external thiamine and is blocked by veratridine, suggesting that it is a self-exchange mechanism catalyzed by the high affinity thiamine transporter. The remaining thiamine efflux is neither sensitive to veratridine nor to Ca2+ and its mechanism is unknown. About 25% of intracellular thiamine is not released, even after treatment of the cells with digitonin, thus maintaining an apparent gradient. This suggests a binding or sequestration in intracellular compartments.(ABSTRACT TRUNCATED AT 250 WORDS)