The mechanisms involved in feedback regulation of type I procollagen synthesis by the N-terminal propeptide of the pro alpha 1(I) chain, termed Col 1, are poorly understood. We have constructed a metallothionein-human collagen chimeric minigene (pMTCol) that codes for a Col 1 fusion protein but lacks a signal peptide sequence and, therefore, would be expected to direct the synthesis of the fusion protein to the cytosol. Baby hamster kidney cells and fetal calf ligament cells, transfected with pMTCol, transcribed the gene and synthesized an intracellular antigen that was identified as the fusion protein with a monospecific antibody. Transfected fetal calf ligament fibroblasts showed significantly reduced levels of endogenously produced type I collagen, as determined by imaging and digital quantitation of immunofluorescence by confocal microscopy; synthesis of fibronectin, thrombospondin, and SPARC (secreted protein, acidic and rich in cysteine) was unchanged or increased in these cells. This recombinant approach offers the potential for a systematic analysis of feedback regulation of collagen synthesis.