Specific [3H]-substance P binding was saturable and of high affinity (KD = 2.5 nM) with a Bmax of 725 fmol/mg protein in the isolated rabbit iris sphincter muscle. The competition for [3H]-substance P binding was in the order of eledoisin greater than substance P greater than kassinin greater than neurokinin B greater than neurokinin A greater than physalaemin. In the same preparation, neurokinin A, as well as substance P induced a concentration-related accumulation of [3H]-inositol phosphates (IPs), and the maximum increase was about 200% of the control at 10(-4) M. [D-Arg1, D-Trp7,9, Leu11]-substance P (SP) and [D-Pro2, D-Trp7,9]-SP (10(-3) M) inhibited substance P or neurokinin A (10(-4) M)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis significantly. [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP (10(-3) M) also inhibited neurokinin A (10(-4) M)-induced PIP2 hydrolysis significantly. Neurokinin A and substance P produced concentration-related contractions in normal Ca(2+)-containing medium. The contractile response was weaker in Ca(2+)-free medium, and there was no response in 0.2 mM EGTA medium. In Ca(2+)-free medium, the basal level of [3H]-IPs accumulation was smaller than that in normal medium, and neurokinin A and substance P significantly increased PIP2 hydrolysis. In the 0.2 mM EGTA containing medium, neurokinin A and substance P did not stimulate the PIP2 hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)