The tachykinin peptide, bufokinin, isolated from the cane toad intestine, is important in intestinal and cardiovascular regulation in the toad. In this study, three tachykinin NK₁-like receptor isoforms, bNK₁-A, bNK₁-B and bNK₁-C, encoding proteins of 309, 390 and 371 amino acids, respectively, were cloned from the toad brain and intestine. These isoforms differ only at the intracellular C-terminus. The bNK₁-A and bNK₁-B isoforms are similar to the truncated and full-length forms of the mammalian NK₁ receptor, whereas bNK₁-C is unique and does not correspond to any previously described receptor. RT-PCR studies demonstrated that three isoform transcripts are widely distributed in the toad with high expression in gut, spinal cord, brain, lung and skeletal muscle. When expressed in COS-7 cells, bufokinin showed similar high affinity (IC₅₀ 0.6-0.8 nM) in competing for [¹²⁵I]Bolton-Hunter bufokinin binding at all receptors, but the binding affinities of substance P (SP) and neurokinin A (NKA) were very different at each isoform. When expressed in Xenopus oocytes, the truncated isoform, bNK₁-A, was inactive, whereas bNK₁-B and bNK₁-C produced changes in chloride current when stimulated by tachykinins (minimum concentrations: bufokinin 0.1 nM, SP 1 nM and NKA 10 nM). A marked desensitization of the response was seen to subsequent applications of tachykinins, as experienced by the mammalian NK₁ receptor. In summary, our study describing three isoforms of NK₁-like receptor from the toad suggests that the alternative splicing of NK₁ receptor is a physiologically conserved mechanism, and raises a fundamental question as to the physiological role of each isoform.