The phosphate analogue vanadate, at 10 mM, strongly (approximately 90%) inhibited the autophagic sequestration of endogenous lactate dehydrogenase in isolated rat hepatocytes. The effect of vanadate was markedly (approximately 80%) antagonized by asparagine (20 mM), and to a lesser extent by glutamine, glycine, and alanine. The antagonism was only observed in the presence of Ca2+ when an isotonic standard incubation medium was used, but by increasing the medium osmolality this Ca2+ requirement could be eliminated. Asparagine induced a cell swelling (17% at 20 mM) that might account for at least part of its vanadate antagonism, since hypotonic cell swelling by itself stimulated autophagy (with a maximal effect at approximately 200 mosM). Conversely, hypertonic media inhibited autophagy and were additive to vanadate. In a strongly hypotonic medium (less than 200 mosM), both asparagine and vanadate were inhibitory. However, since vanadate alone had no effect on cell volume, the vanadate-asparagine antagonism could not be exerted exclusively at the level of cell volume regulation. An additional mechanism might be a partial deamination of asparagine, generating ammonia, which was found to oppose the vanadate inhibition of autophagy while having no effect on cell volume. Other metabolizable amino acids, like alanine and glycine, were moderately vanadate-antagonistic while failing to induce cell swelling. These results are compatible with a vanadate-antagonistic effect of asparagine mediated partly through an unknown mechanism (possibly pH change) by its deamination product, ammonia, partly through cell swelling and a secondary Ca2+ influx that could compensate for a vanadate-induced depletion of intracellular calcium stores.