Vesicles have been prepared from Ehrlich cells by using a method based on the early experiments of Forte et al. (Forte, J.G., Forte, T.M. and Heinz, E. (1973) Biochim. Biophys. Acta 298, 827-841) with some minor modifications, using the filter technique. From electron micrographs and from the sensitivity of these vesicles towards osmotic pressure changes in the medium induced by (nonpermeant) sucrose, it is concluded that the vesicles are closed. The counterflow phenomenon with glycine and Na+-linked contransport of glycine appears to indicate that these vesicles are still functioning. The observation of the overshoot phenomenon interpreted in terms of theoretical predictions confirms that the active accumulation of glycine is energized by the Na+ electrochemical potential gradient. In particular, the contribution of the electrical components of these gradients is evidenced by the effects of the anion of the added sodium salt or of the addition of valinomycin. In contrrast to observations by others we found that ouabain does not directly affect Na+-linked cotransport of glycine whereas HgCl2 does so. Nor could any significant overshoot be demonstrated in the absence of an Na gradient. Since these vesicles were not metabolically active, these experiments do not exclude the possibility that in intact cells glycine is in addition transported primarily or partially actively.