1. The potency of the non-peptide antagonists CP-96,345 and RP 67580 on NK1 receptor-stimulated [3H]-inositol phosphate accumulation in cell lines or tissue from three different species has been examined. 2. We have used: UC11 cells, derived from a human astrocytoma, and rat LRM55 glial cells, both of which express large numbers of functional NK1 receptors, and the well characterized guinea-pig ileum which expresses both NK1 and NK3 receptors. 3. RP 67580 has an approximately 25 fold lower affinity for NK1 receptors in human UC11 cells (Kd = 194 nM) than in rat LRM55 cells (Kd = 7.9 nM), in contrast CP-96,345 has an approximately 200 fold lower affinity in rat LRM55 cells (Kd = 210 nM) relative to human UC11 cells (Kd = 0.99 nM). The pharmacological profile of CP-96,345 and RP 67580 in guinea-pig ileum was similar to that observed in human UC11 cells. 4. In conclusion, we have demonstrated that previously reported species differences in binding affinities for the non-peptide NK1 antagonists, CP-96,345 and RP 67580, are also observed in inhibition of NK1 receptor stimulated hydrolysis of inositol phospholipids.