Tachykinins such as substance P (SP) and neurokinin A (NKA) acting on neurokinin (NK) receptors modulate the nonadrenergic noncholinergic (NANC) neurotransmission in the gastrointestinal tract of several species, but the information about the mouse small intestine is scanty. Both SP and NKA induced concentration-dependent contractions of ileal segments isolated from wild-type mice that were blocked by NK₁ and NK₂ antagonists, respectively. In contrast, segments isolated from NK₁ receptor (NK₁-R) knockout mice responded only to elevated concentrations of SP. To reveal the inhibitory NANC (iNANC) responses, tissues were pretreated with atropine and guanethidine. Under these conditions, a tetrodotoxin-sensitive relaxation in response to electrical field stimulation (EFS) was observed. NK₁-R knockout mice presented a trend toward an increase in iNANC responses, whereas the NK₁-R antagonist significantly potentiated iNANC relaxation in tissues isolated from wild-type mice. N^G-nitro-L-arginine methyl ester (100 µM) transformed the relaxant response to EFS into a tetrodotoxin-sensitive, frequency-dependent contraction characteristic of an excitatory NANC (eNANC) system. A NK₁-R antagonist abolished the contractile responses of the mouse ileum to EFS, whereas a NK₂ receptor antagonist had a trend toward reducing EFS-induced contraction. The eNANC component was absent in NK₁-R knockout mice. Measurement of SP-like immunoreactivity indicated similar amounts of SP per gram of tissue isolated from wild-type and NK₁-R knockout mice, indicating that the observed differences in response to EFS were not due to a differential peptide content. It is concluded that, in the mouse ileum, both NK₁ and NK₂ receptors modulated the responses to exogenous tachykinins, whereas NK₁ is the primary tachykinin receptor involved in both iNANC and eNANC transmission.