Type I collagen is the main constituent of extracellular matrix found in various organs including the heart. Under some pathological conditions accumulation of excess type I collagen in the interstitium leads to organ dysfunction. In order to identify the regulatory elements in the rat alpha 1(I) collagen gene promoter, deletions were made in the promoter region. Various plasmid constructs were transfected into different fibroblasts using LipofectAMINE. The results indicated a negative cis-element between nucleotides -310 to -440 in the rat alpha 1(I) collagen gene promoter. Presence of this sequence significantly diminished the reporter gene activity. In addition we have observed that the sequence between -220 to -330 contained a positively acting cis-element, which is highly active in rat fibroblasts. Analysis of the nuclear factors binding to the negative element by electrophoretic mobility shift assays indicated that similar or identical factors are present in different fibroblasts as well as human HeLa cells and that these factors appear to bind to a composite sequence within -325 to -400. Competition with different oligonucleotides suggested that two distinct but contiguous sequence motifs may constitute the negative regulatory element. Our results with the rat alpha 1(I) collagen promoter confirm the presence of a negative cis-element previously described for the mouse promoter and provided additional information on the bipartite nature of this element.