Cells were isolated from pig gastric mucosa by a combination of mechanical and enzymatic treatment. Isopycnic centrifugation on linear density gradients of Percoll provided a simple and rapid procedure for obtaining highly enriched parietal cells and chief cells. Their densities were 1.06 and 1.10 g/ml, respectively. Isolated mucosal cells attached to Petri dishes coated with fibronectin or collagen. Both parietal cells and chief cells adhered more readily to fibronectin than collagen. Mucosal cells and cells from the Percoll gradients were maintained for up to one week as primary cell cultures. The ability of free parietal cells to produce acid was tested by the 14C-aminopyrine accumulation technique. Maximal accumulation was 2.5 pmol aminopyrine per 10(4) parietal cells after incubation for 45 min at 10(-4) M histamine. The EC50 for histamine was 5 X 10(-6) M. The accumulation of aminopyrine at 10(-6) M carbachol and 10(-7) M pentagastrin were for both secretagogues about 0.9 pmol per 10(4) parietal cells.