The effect of two cardiac sarcolemmal inhibitory peptides, the 20-amino acid exchange inhibitory peptide (XIP) and the molluscan cardioexcitatory tetrapeptide amide Phe-Met-Arg-Phe-NH2 (FMRFa), were tested in dialyzed squid giant axons. XIP injected into axons causes a maximal inhibition of 52 +/- 8% (n = 6) in the external Na (Nao)-dependent Ca efflux. The inhibitory effect was the same in axons dialyzed with saturating intracellular Ca (Cai) concentration (100 microM) and no MgATP or in axons containing submicromolar Cai concentrations (0.7 microM) and 2 mM MgATP. FMRFa, a peptide that shows no obvious homology with XIP, also causes a marked inhibition in Nao-dependent Ca efflux. As in cardiac sarcolemmal vesicles, the peptide inhibits with low apparent affinity (Ki = 1.9 microM; n = 5). Like XIP, FMRFa has the same effect in axons dialyzed with or without MgATP. The data indicate that XIP, which resembles an endogenous calmodulin binding site that may have an autoregulatory function, and the tetrapeptide FM-RFa, which binds to a putative opiate site, both inhibit Na/Ca exchange in squid axons. The sites at which these peptides bind are not related to the nucleotide (MgATP) regulation of Na/Ca exchange. We therefore suggest that these two sites in the vertebrate cardiac Na/Ca exchange are conserved in the invertebrate axon exchanger.