Binding experiments performed with [I-125]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK1 receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK, but neither NK2 nor NK3 receptors. In all cases, [I-125]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [125I]-NKA binding sites were notably different from those of classical NK1 binding sites selectively labelled with [I-125]-BHSp. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [I-125]-NKA than [I-125]-BHSP binding sites. Interestingly, differences were also found in the ratio of B-max values for [I-125]-NKA and [I-125]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK, binding sites are present on distinct NK1 receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations,