The activity of the exogenous, full-length IGFII P3 promoter is significantly up-regulated during the logarithmic growth phase, but rapidly declines in confluent CaCo2 cells undergoing differentiation. Nuclear run-on assays confirmed cell-density-dependent regulation of endogenous P3 promoter. In order to identify regulatory elements in the P3 promoter that may be required for regulating cell-density-dependent transcriptional activity, we used the methods of promoter truncation, EMSA, DNase footprinting and mutation analysis. The relative activity of the full-length (-1229/+140) and truncated (-1090/+140) promoter was identical, being ~ 19, 27, 7 and 3 % of pSV-luc activity on Days 3, 5, 7 and 9 of cell culture, respectively. However, truncation to ?1048 resulted in complete loss of cell-density-dependent down-regulation of P3 promoter activity on Days 7 and 9, suggesting the presence of regulatory elements between ?1091/-1048 sequence. Further step-wise truncation to ?515 did not change promoter activity. Truncation to ?138/+140 resulted in complete loss of promoter activity, suggesting that the core promoter was within ?515/?138 segment. A 14 bp footprint (-1084/-1070) was identified by DNase footprinting within the distal -1091/-1048 segment. EMSA with wild type ( wt ) and mutant (mut) probes confirmed the presence of a novel 7 bp (CGAGGGC)(-1084/-10 78) cis element (P3-D); its mutation abolished binding. Functionality of P3-D cis element was confirmed by measuring activity of core P3 promoter ligated to distal P3 segment containing either the mut or wt P3-D element. We have, therefore, identified a novel cis element, P3-D, that appears to play a critical role in regulating IGF-II P3 promoter activity in a cell-density/differentiation-dependent manner.