Uptake and metabolism of L-carnitine, D-carnitine and acetyl-L-carnitine were studied utilizing isolated guinea-pig enterocytes. Uptake of the D- and L-isomers of carnitine was temperature dependent. Uptake of L-[14C]carnitine by jejunal cells was sodium dependent since replacement by lithium, potassium or choline greatly reduced uptake. L- and D-carnitine developed intracellular to extracellular concentration gradients for total carnitine (free plus acetylated) of 2.7 and 1.4, respectively. However, acetylation of L-carnitine accounted almost entirely for the difference between uptake of L- and D-carnitine. About 60% of the intracellular label was acetyl-L-carnitine after 30 min, and the remainder was free L-carnitine. No other products were observed. D-Carnitine was not metabolized. Acetyl-L-carnitine was deacetylated during or immediately after uptake into intestinal cells and a portion of this newly formed intracellular free carnitine was apparently reacetylated. L-Carnitine and D-carnitine transport (after adjustment for metabolism and diffusion) were evaluated over a concentration range of 2-1000 microM. Km values of 6-7 microM and 5 microM, were estimated for L- and D-carnitine, respectively. Ileal-cell uptake was about half that found for jejunal cells, but the labeled intracellular acetylcarnitine-to-carnitine ratios were similar for both cell populations. Carnitine transport by guinea-pig enterocytes demonstrate characteristics of a carrier-mediated process since it was inhibited by D-carnitine and trimethylaminobutyrate, as well as being temperature and concentration dependent. The process appears to be facilitated diffusion rather than active transport since L-carnitine did not develop a significant concentration gradient, and was unaffected by ouabain or actinomycin A.