Apical plasma membrane vesicles were prepared from human organ donor colon mucosal scrapings. These vesicles were enriched 10-fold in cysteine-sensitive alkaline phosphatase activity compared to starting homogenates, and showed minimal contamination of microsomal, mitochondrial or basolateral membranes. Transport studies using [22Na] uptake into proximal colonic vesicles demonstrated Na+ and H+ conductances, Na+/H+ exchange and amiloride inhibition of Na+ uptake. The isolation of these apical vesicles will permit detailed study of human colonic transport processes.