Neuronal differentiation and neurite outgrowth are key processes during development of the nervous system. Understanding the regulation of neurite outgrowth stimulated by neurotrophins is crucial to developing therapies to promote axon regeneration after injury or in neurodegenerative diseases. Treatment of PC12 cells with nerve growth factor (NGF) stimulates them to extend neurites and differentiate into a sympathetic neuron-like phenotype. In this study we found that exposure of PC12 cells to 42 degrees C for 1 h significantly enhanced NGF-induced neurite elongation, but not branching. This heat shock treatment led to induction of heat shock protein 25 (Hsp25) and Hsp70. The morphological changes induced by NGF were accompanied by increased Hsp25 mRNA levels, in addition to elevation in Hsp25 protein expression and phosphorylation, without a concomitant increase in Hsp70. A possible role for Hsp25 in NGF-stimulated neurite outgrowth was investigated. However, quantification of NGF-induced neurite elongation and branching revealed that neither of these features were altered in PC12 cells which stably overexpressed human Hsp27 (to mimic heat shock induction of Hsp25). Similarly, knockdown of Hsp25 using siRNA had no effect on NGF-induced neurite outgrowth. Inhibition of p38 MAPK signalling with SB202190 blocked phosphorylation of Hsp25 without affecting NGF-induced neurite outgrowth or the heat shock-dependent enhancement of elongation. These findings indicate that Hsp25 is not required for NGF-induced neurite outgrowth in PC12 cells and is not responsible for the heat shock-enhancement of NGF-induced neurite elongation. Instead, inhibition of MEK1/2 with U0126 partially reduced the heat shock-enhancement of NGF-stimulated neurite elongation.