Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. However, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. In the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdAd) in comparison with a first generation vector. Our results demonstrate that the hdAd system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. Neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. This was followed by an increase in the fraction of TUNEL-positive cells and a loss of neuronal survival. In contrast, hdAds could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability. Gene Therapy (2000) 7, 1200-1209.