Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca²⁺ imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10⁻⁶ M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K⁺-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10⁻⁶ M and CRF 10⁻⁷ M during 30 s induced a Ca²⁺ response in 22.4% of the myenteric neurons ( n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin ( n = 55). The selective CRF-1 receptor antagonist CP 154,526 ( n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10⁻⁷ M, and its effect at 10⁻⁶ M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10⁻⁶ M ( n = 70) could not block the CRF-induced Ca²⁺ transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca²⁺ blocked all responses to CRF ( n = 47). L-type channels did not contribute to the CRF-induced Ca²⁺ transients. Blocking N- or P/Q-type Ca²⁺ channels did not reduce the responses significantly. Combined L- and R-type Ca²⁺ channel blocking (SNX-482 10⁻⁸ M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca²⁺ transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca²⁺ transients highly depend on somatic calcium influx through voltage-operated Ca²⁺ channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca²⁺ signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.