5-Hydroxytryptamine (5-HT) has been reported to produce significant responses in blowfly salivary glands, but little information is available concerning its action on mammalian salivary glands. When 5-HT (0.1 mumol/L to 10 mumol/L) is infused i.a. into anesthetized rats, no salivary secretion is obtained from either parotid or submandibular glands. However, when 5-HT is infused along with a threshold concentration of acetylcholine (0.1 mmol/L), potentiation of parotid secretory response is seen with 5-HT (1 mumol/L, 260% increase; 10 mumol/L, 146% increase). Substance P (0.3 mumol/L) combined with 5-HT (1 mumol/L) also resulted in a potentiation of parotid secretion (160% increase). Protein and calcium concentrations were not altered during such treatments. No potentiation of submandibular secretion was noted. Experiments in vitro with parotid cell aggregates exhibited no potentiation associated with the combined use of 5-HT and carbachol, as measured by amylase secretion and inositol trisphosphate accumulation. The experiments indicate that 5-HT substantially modulates parotid salivary secretion in vivo; however, the in vitro findings suggest that 5-HT does not act directly on surface glandular receptors. The magnitude of the in vivo potentiation could very well implicate circulating or released 5-HT as a physiological modulator of endogenous neurotransmitter action.