This study reports the functional expression of a specific, high affinity carrier-mediated mechanism for transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. Initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH-, temperature-, and energy- dependent; it was, however, Na-independent in nature. Evidence for the existence of a carrier-mediated system that is specific for 3H -nicotinic acid transport were found and included: 1) saturability as a function of concentration with an apparent Km and Vmax of 0.73 + 0.16 μM and 25.02 + 1.45 pmol/mg protein/3 min, respectively; 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, valproate), and 3) trans-stimulation of 3H-nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca2+ -calmodulin -mediated pathway (but not modulators of the PKC, PKA and PTK-mediated pathways) inhibit nicotinic acid transport into both HepG2 cells and in human primary hepatocytes. Maintaining HepG2 cells (for 48 hrs) in a growth medium over-supplemented with nicotinic acid (or nicotinamide) did not affect subsequent transport of 3H-nicotinic acid into HepG2 cells. These results show for the first time the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.