Short-chain fatty acids are the predominant luminal anion in the colon and are generally absorbed rapidly in vivo. However, the mechanisms of in vitro transport of short-chain fatty acids have not been fully delineated. Therefore, we examined [14C]propionate fluxes in rabbit proximal colon under short-circuit conditions. There was minimal metabolism of propionate (less than 10%), permitting accurate flux measurements using a radioisotopic tracer. In a 20 mmol/L propionate Ringer's solution at pH 7.4, there was a significant rate of propionate secretion (-0.58 +/- 0.08 microEq.cm-2.h-1). Decreasing pH to 6.8 by decreasing bicarbonate concentration in the bathing medium resulted in increases in unidirectional fluxes but no change in net transport. Reversal of propionate secretion to propionate absorption was elicited by HEPES substitution for bicarbonate at pH 6.8 or by serosal addition of epinephrine, which increases apical Na(+)-H+ exchange in this epithelium. Propionate absorption was blocked by both amiloride, an Na(+)-H+ exchange inhibitor, and ouabain. Under basal conditions, there was a concentration-dependent increase in basal unidirectional propionate fluxes with no change in net transport as the concentration of propionate increased from 10 to 60 mmol/L. In contrast, a concentration-dependent saturation of epinephrine-stimulated propionate absorption was apparent. Transepithelial propionate gradients did not yield a significant diffusion potential. These results suggest that, in rabbit proximal colon, (a) there is bidirectional diffusion of propionate, most probably in the protonated rather than the ionized form; (b) a component of propionate transport is active and linked to electroneutral Na+ absorption through apical Na(+)-H+ exchange; and (c) changes in composition of the fluid bathing the proximal colon in vitro may significantly alter both rates and direction of short-chain fatty acid transport. Regulation of transcellular active transport may play an important role in colonic short-chain fatty acid conservation.