The isolation and enrichment of the gastric chief cells of the rat are described. Ultrastructural examination showed 85% enrichment from a mixed population of mucosal cells following their centrifugation through a discontinuous Percoll gradient. When compared to homogenates of the initial mixed cell population, the enriched chief cell population showed over a three-fold increase in pepsin(ogen) content. Preliminary experiments showed that a combination of the secretagogues histamine and carbamylcholine caused a significant increase in pepsin release from enriched chief cell preparations and a concomitant decrease in their pepsin content as compared to untreated cells. The results obtained in this study indicate the feasibility of employing this procedure for the isolation of gastric chief cells for the in vitro study of secretagogue regulation of pepsin secretion.