The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.