N-myristoyltransferases (NMT) add myristate to the NH(2) termini of certain proteins, thereby regulating their localization and/or biological function. Using RNA interference, this study functionally characterizes the two NMT isozymes in human cells. Unique small interfering RNAs (siRNA) for each isozyme were designed and shown to decrease NMT1 or NMT2 protein levels by at least 90%. Ablation of NMT1 inhibited cell replication associated with a loss of activation of c-Src and its target FAK as well as reduction of signaling through the c-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays showed that depletion of either NMT isozyme induced apoptosis, with NMT2 having a 2.5-fold greater effect than NMT1. Western blot analyses revealed that loss of NMT2 shifted the expression of the BCL family of proteins toward apoptosis. Finally, intratumoral injection of siRNA for NMT1 or for both NMT1 and NMT2 inhibited tumor growth in vivo, whereas the same treatment with siRNA for NMT2 or negative control siRNA did not. Overall, the data indicate that NMT1 and NMT2 have only partially overlapping functions and that NMT1 is critical for tumor cell proliferation.