The efflux of l-[ 3H]carnitine was studied in cells from an established cell line from human heart (Girardi human heart cells, CCL 27). The cells were loaded with 4 μmol/l l-[ 3H]carnitine for 1 or 24 h, and the efflux of radioactivity into the medium was measured. The amount of intracellular l-[ 3H]carnitine retained was expressed as a function of time. The results were fitted to an exponential equation, from which efflux rate constants were computed. Increasing the extracellular concentration of butyrobetaine, l-carnitine, d-carnitine, betaine, dl-norcarnitine or 3-dimethylamino-2-hydroxypropionic acid each increased the observed efflux. This is most likely due to accelerated exchange diffusion. The substrate specificity of this accelerated exchange diffusion is different from what previously has been found in competitive uptake studies of l-carnitine. l-Carnitine was preferentially released to l-acetylcarnitine, and blocking the sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) increased the efflux.