To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.