In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.