1. Opioid peptides promote net intestinal absorption via two mechanisms: stimulation of Na+ and Cl‐ absorption and inhibition of Cl‐ secretion. Although these transport changes are predominantly mediated by submucosal neurones, it is currently unclear whether opioid peptides can regulate enterocyte function directly. We therefore tested the hypothesis that enterocytes have specific opioid receptors. 2. Villus and crypt jejunal epithelial cells were isolated by the distended sac method from anaesthetized guinea‐pigs. Flow cytometry was used to resolve enterocytes from other cell types and to determine whether binding of a fluorescently labelled opioid antagonist, naltrexone‐FITC, could be prevented by unlabelled mu‐ and delta‐opioid receptor agonists. A population of crypt enterocytes (approximately 21%) exhibited high‐affinity naltrexone‐FITC binding to both mu‐ and delta‐type binding sites that was stereoselective and sodium dependent. Villus enterocytes did not exhibit any of these characteristics. 3. Basal cAMP production was elevated in both villus and crypt cells treated with IBMX (3‐isobutyl‐1‐methylxanthine). Villus cells did not respond to 100 nM vasoactive intestinal peptide (VIP), nor were they affected by opioid peptides. In contrast, 100 nM VIP significantly increased cAMP production in crypt epithelial cells, which was significantly reduced by both morphiceptin and D‐Ser2‐Leu‐Enk‐Thr. This opioid‐mediated effect was stereoselective and blocked by the opioid receptor antagonist naltrexone. 4. These experiments suggest that enterocytes isolated from the crypt epithelium of guineapigs have both mu‐ and delta‐types of opioid receptors. It is possible that these cells participate in opioid‐mediated regulation of intestinal secretion.