Dimeric rabbit muscle creatine kinase (MM-CK) was bound to CNBr-activated Sepharose 4B by one of its subunits (MM-CKA). Treatment of MM-CKA with guanidine hydrochloride released the unbound subunit to yield the matrix-bound monomer (M-CKB). M-CKB recombined with dissociated MM-CK soluble subunits to reconstitute a matrix-bound dimer (MM-CKC). M-CKB also associated with dissociated subunits of BB-CK from crude extracts of rabbit brain and of arginine kinase from sea cucumber muscle (MM-AK) to form the matrix-bound heterohybrids MB-CKC and M-CK/M-AKC, respectively. Guanidine hydrochloride gradient elution studies showed that MM-CKA, MM-CKC and MB-CKC were all dissociated at the same concentration of the denaturant (0.96 M), while the M-CK/M-AKC heterohybrid was less stable, dissociating at 0.5 M. The specific interaction between subunits of echinoderm and mammalian phosphagen kinases to form a hybrid enzyme of dual substrate specificity supports the view that these enzymes had a common evolutionary origin.