RasGRP is a recently described guanine nucleotide exchange factor (GEF) that possesses a single C1 domain homologous to that of protein kinase C (PKC). The phorbol ester [(3)H]phorbol 12, 13-dibutyrate ([(3)H]PDBu) bound to this C1 domain (C1-RasGRP) with a dissociation constant of 0.58 +/- 0.08 nM, similar to that observed previously for PKC. Likewise, the potent PKC activator bryostatin 1, a compound currently in clinical trials, showed high affinity binding for C1-RasGRP. Structure activity analysis using several phorbol ester analogs showed both similarities and differences in ligand selectivity compared with PKC; the differences were comparable in magnitude to those between different PKC isoforms. Similarly, the potency of the PKC inhibitor calphostin C to inhibit [(3)H]PDBu binding to C1-RasGRP was similar to that observed for PKC. In contrast to the relative similarities in ligand recognition, the lipid cofactor requirements differed between RasGRP and PKC. The C1 domain plus the EF-hand motif of RasGRP (C1EF-RasGRP) was markedly less dependent on acidic phospholipids than was PKCalpha. The differences in lipid requirements were reflected in differential ligand selectivity under conditions of limiting lipid. Despite the presence of twin EF-hand like motifs, calcium did not affect the binding of [(3)H]PDBu to C1EF-RasGRP. We conclude that RasGRP is a high affinity receptor for phorbol esters and diacylglycerol. RasGRP thus provides a direct link between diacylglycerol generation or phorbol ester/bryostatin treatment and Ras activation.