Neuropeptide Y (NPY), an orexigenic peptide synthesized in-vivo within the arcuate nucleus and released in the paraventricular nucleus, causes hyperphagia and anxiolysis. To determine the presence of NPY synthesis and secretion by fetal rat neural cells that were immortalized by the temperature sensitive large T antigen, we undertook the present study. Using undifferentiated (33°C, serum-containing medium) and differentiated(39°C, serum-free defined medium) neural cells predominantly expressing neuronal (neurofilament [NF] positive, glial fibrillary acidic protein [GFAP] negative), and glial (NF -ve, GFAP +ve) phenotypes, we characterized the presence of NPY mRNA by Northern blot analysis (n=2), cellular peptide by immunohistochemical analysis (n=4), and extracellular secretion of the NPY peptide over 20 min by radioimmunoassay of Kreb's buffer conditioned medium(n=10). Differentiation of neuronal and glial enriched cell cultures led to a 24-fold and 13-fold increase in NPY mRNA (0.8 kb) concentrations respectively(p <0.01). A similar change was noted in the number of cells expressing NPY immunoreactivity, this immunoreactivity being noted only in well differentiated neuronal cells and their processes. The increase in NPY immunoreactive cell number with differentiation was not paralleled by an increase in the extracellularly secreted NPY peptide levels. Employing 10μm of antisense oligonucleotide complementary to 20 bp downstream starting from the ATG-translation start site codon of the rat preproNPY gene, the number of NPY immunoreactive neuronal cells diminished 6-fold when compared to corresponding sense and missense oligonucleotide treated cells (n=3 each; p.<0.05). We conclude that 1] immortalized fetal neuronal cells synthesize and actively secrete NPY, the rate of synthesis being higher in differentiated cells, and 2] appropriately synthesized antisense oligonucleotides suppress neuronal NPY peptide production. These in-vitro studies will set the stage for undertaking in-vivo selective neuronal knock out of the hypothalamic NPY, thereby allowing phenotypic characterization of postnatal animals in the absence of hypothalamic NPY production.