Extracellular signal-regulated protein kinase 5 (ERK5) is a recently discovered orphan mitogen-activated protein kinase for which no substrates or strong activators have been described. Two ERK5 chimeras were created as a novel approach to discover its substrates and upstream regulators. One chimeric protein contained the N-terminal domain of the ERK5 catalytic core (subdomains I-IV) and the C-terminal domain of the ERK2 catalytic core (subdomains V-XI). This chimera was highly responsive to stimuli that regulate ERK2 in vitro and in cells. A second chimeric protein consisted of the N-terminal domain of ERK2 (subdomains I-IV) and the C-terminal domain of the ERK5 catalytic core (subdomains V-XI). This chimera was activated in bacteria by coexpression with a constitutively active mutant of MEK1. Using the activated chimera, we identified three in vitro substrates of ERK5. Assaying ERK5 activity in immune complexes with one of these substrates, c-Myc, we determined that the ERK5 catalytic domain is activated by V12 H-Ras and to a lesser extent by phorbol ester but not by constitutively active mutants of Raf-1. Thus, ERK5 is a target of a novel Ras effector pathway that may communicate with c-Myc.