Studies demonstrate that some colon cancers possess receptors for various gastrointestinal hormones or neurotransmitters, the occupation of which can affect growth. These results are limited because frequently only a small number of tumors are studied, only 1 or 2 receptors are sought, and the effect on cell function is not investigated. In the present study, 10 recently characterized human colon cancer cell lines were studied to determine whether they possess receptors for any of 12 different gastrointestinal hormones or neurotransmitters and to determine whether these receptors mediate changes in cellular function. Each of the cell lines exhibited receptors for at least one radioligand. Receptors for vasoactive intestinal peptide (VIP) and muscarinic cholinergic agents occurred on 60%, bombesin and gastrin on 30%, β-adrenergic agents and gastrin-releasing peptide (GRP) on 20%, and somatostatin, opiates, neuromedin B, and substance P on 10%. Analysis of [³H]N-methylscopolamine binding revealed a K_d of 0.2 nm for N-methylscopolamine with a binding capacity of 2500 sites/cell. With the agonist carbamylcholine, the receptor exhibited 2 classes of binding sites: one of high affinity (K_d 55 µm) representing 75% of the binding sites and one of low affinity (K_d 0.3 mm) representing 25% of the binding sites. Analysis of ¹²⁵I-[Tyr⁴] bombesin binding revealed a receptor of high affinity (K_d 2.1 µm) with a binding capacity of 3300 sites/cell. Inhibition of binding by agonists revealed relative potencies of ¹²⁵I-[Tyr⁴]bombesin > GRP >> neuromedin B, and two recently described antagonists were similar in potency to GRP. Analysis of ¹²⁵I-VIP binding revealed a receptor having 2 classes of binding sites: one of high affinity (K_d 3.6 nm) and one of low affinity (K_d 1.7 µm) which represented the majority of the 5.5 × 10⁶ binding sites/cell. The relative potencies of agonists were VIP > helodermin > peptide histidine methionine > secretin. Evaluation of biological activity mediated by the muscarinic cholinergic and bombesin receptors revealed an increase of intracellular calcium and of inositol triphosphate by specific receptor agonists. The presence or absence of receptors detected by binding correlated closely with the ability of selective receptor agonists to alter cell function. These results demonstrate the presence of several different receptors for gastrointestinal hormones or neurotransmitters, some described for the first time, on human colon cancer cell lines, including bombesin-related peptides, VIP, somatostatin, substance P, β-adrenergic agents, calcitonin gene-related peptide, gastrin, muscarinic cholinergic agents, and opiates. These receptors are functional because occupation by selective agonists altered intracellular mediators. These results suggest that it will be important to extend these studies to evaluate growth effects.