Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms.