Caspase-9 plays a critical role in the initiation of apoptosis by the mitochondrial pathway. Activation of caspase-9 is inhibited by phosphorylation at Thr125 by ERK1/2 mitogen activated protein (MAP) kinases in response to growth factors. Here, we show that phosphorylation of this site is specific for these classical MAP kinases and is not strongly induced when JNK and p38a/ß MAP kinases are activated by anisomycin. By deletion and mutagenic analysis, we identify domains in caspase-9 and ERK2 that mediate their interaction. Binding of ERK2 to caspase-9 and subsequent phosphorylation of caspase-9 requires a basic docking domain (D domain) in the N–terminal prodomain of the caspase. Mutational analysis of ERK2 reveals a 157TTCD160 motif required for recognition of caspase-9 that acts independently of the putative common docking (CD) domain. Molecular modelling supports the conclusion that Arg10 in the D domain of caspase-9 interacts with Asp160 in the TTCD motif of ERK2. Differences in the TTCD motif in other MAP kinase family members could account for the selective recognition of caspase-9 by ERK1/2. This selectivity may be important for the anti-apoptotic role of classical MAP kinases in contrast to the pro-apoptotic roles of stress-activated MAP kinases.