Excess neurons in the developing nervous system are eliminated by apoptosis, an ordered cascade of proteolytic events orchestrated by the caspase family of proteases. The apoptotic machinery is tightly regulated by a variety of extracellular signals that either activate or suppress apoptosis after binding to receptors on neurons. These signals are integrated in neurons by a complex network of protein-protein interactions that bring about transcriptional and posttranslational changes in key regulators of the apoptotic machinery; such regulators include members of the Bcl-2 family. Homeodomain-interacting protein kinase-2 (HIPK2) is a recently identified nuclear serine-threonine kinase that interacts with homeodomain transcription factors and participates in the regulation of cell growth and genotoxic stress-induced apoptosis. Here we show that overexpression of HIPK2 in developing neurotrophin-dependent sensory and sympathetic neurons promotes apoptosis of these neurons grown with neurotrophins. HIPK2-induced apoptosis is caspase-dependent, is inhibited by overexpression of Bcl-2 and Bcl-W, and fails to occur in Bax-deficient neurons. Trigeminal sensory neurons, which are especially susceptible to HIPK2-induced apoptosis, express the highest levels of HIPK2 during the peak of apoptosis in vivo. Knockdown of endogenous HIPK2 with antisense oligonucleotides substantially reduces and delays apoptosis after neurotrophin deprivation in vitro. These findings identify HIPK2 as a novel participant in programmed cell death in the developing peripheral nervous system.