The contraction of the longitudinal muscle of the guinea-pig isolated ileum in response to substance P (SP) in high [K+] medium and in Ca2+-free solution which contained EGTA has been investigated in order to examine whether excitation-contraction coupling involves the release of Ca2+ from an intracellular store. In tissues contracted by K+, high concentrations of SP (greater than or equal to 0.1 microM) were still capable of causing a slight, transient contraction. Bathing the ileum in a Ca2+-free medium for 2.5 min greatly diminished the potency and efficacy of SP in contracting the longitudinal muscle but concentrations of 2.2-22 microM SP were still able to produce a response 30-40% of maximal. The responsiveness to SP was completely lost within 10 min of bathing in Ca2+-free medium but was partially restored by a brief exposure (0.5-2 min) to high concentrations of Ca2+ (9-72 mM). The restorative effect of Ca2+ depended on the concentration of Ca2+ and on the time for which the tissue was exposed to this Ca2+ concentration. The fact that high concentrations of SP were able to elicit a contraction in media containing high [K+] or no Ca2+, suggested that they may do so by releasing Ca2+ from an intracellular store which is not as sensitive to removal of extracellular Ca2+ or as easily accessible to EGTA as the extracellular space of the muscle. The location of this store is not known; it may be associated with the internal side of the cell membrane. There is apparently an overlap between the Ca2+ pool associated with the action of SP and the Ca2+ pools utilized by acetylcholine, histamine or tetraethylammonium, which accounts for the cross-desensitization observed between these agonists. It was not possible to determine whether autodesensitization to SP also results from depletion of an intracellular Ca2+ store.