Adult pancreatic ductal cells are believed to be islet precursors. Our aim was to obtain an enriched human ductal cell population in defined culture conditions, and to characterize these cultures for the presence of pancreatic developmental transcription factors. Non-endocrine adult human pancreatic digest was cultured for 4 days in serum-containing and serum-free media. During this time, analysis was done for phenotypic changes, cell death, and expression of islet and islet precursor markers. Culture in serum-supplemented and serum-free media gave similar recoveries of an enriched ductal population after 4 days. Extensive cell death due to apoptosis and necrosis was also observed over this time period. A donor-age dependent expression of pancreatic and duodenal homeobox gene-1 (PDX-1) in ductal cells was seen at 4 days whereby donors <25 yr expressed significantly more than donors >25 yr. Analysis of gene expression by RT-PCR showed the presence of islet developmental transcription factors neuroD, Nkx6.1, and PDX-1, as well as mature islet hormones. While acinar-ductal transdifferentiation of some cells cannot be ruled out, we provide evidence that the predominant mechanism for the derivation of enriched human ductal cultures in our culture conditions is selective acinar cell death. Furthermore, we have shown that ductal cultures from younger donors exhibit greater plasticity through expression of PDX-1, and may be of greater value in attempts to induce islet neogenesis. The presence, however, of insulin and glucagon mRNA indicates that contaminating endocrine cells remain in these cultures and underscores the need to use caution when assessing differentiation potential.