The PCK gene, encoding cytosolic phosphoenolpyruvate carboxykinase, is specifically expressed in gluconeogenic tissues, liver and kidney. Hence it serves as a model of a class of single-copy genes whose transcription is restricted to a few tissues, rather than a unique tissue. To begin delineating the mechanisms that govern this pattern of expression, cis-regulatory elements of PCK were examined using transient transfection assays in PCK-expressing kidney and hepatoma cell lines. The analyses enabled us to identify a proximal element, between nucleotide (nt) positions -121 and -98, relative to the transcription start point that is sufficient for specific expression in kidney cells, but is just one of the elements required for expression in hepatoma cells. A distal element (between nt -487 and -417), which is essential for hepatoma-specific expression, is not needed in kidney cells. We suggest that the differential regulation of PCK expression in the liver and kidney results from an interplay between different cis-regulatory elements and trans-acting factors.