Activation of hepatic stellate cells (HSCs) and other resident mesenchymal cells into myofibroblasts expressing alpha smooth muscle actin (αSMA) and collagen I is a key event in liver fibrogenesis. However, the temporal expression profiles of αSMA and collagen I genes in these cells is unknown. To address this question, we studied αSMA and collagen α1(I) transcriptional patterns in primary cultures of HSCs, and additionally, in an in vivo model of secondary biliary fibrosis using transgenic mice that express the Discomsoma sp. red fluorescent protein (RFP) and the enhanced green fluorescent protein (EGFP) reporter genes under direction of the mouse αSMA and collagen α1(I) promoter/enhancers, respectively. The αSMA‐RFP mice were crossed with collagen‐EGFP mice to generate double transgenic mice. Reporter gene expression in cultured HSCs demonstrated that both transgenes were induced at day 3 with continued expression through day 14. Interestingly, αSMA and collagen α1(I) transgenes were not coexpressed in all cells. Flow cytometry analysis showed three different patterns of gene expression: αSMA‐RFP positive cells, collagen‐EGFP positive cells, and cells expressing both transgenes. αSMA‐only and αSMA/collagen expressing cells showed higher expression levels of synaptophysin, reelin, MMP13, TIMP1, and ICAM‐1 compared to collagen‐only expressing cells, as assessed by real‐time PCR. Following bile duct ligation, αSMA and collagen α1(I) transgenes were differentially expressed by peribiliary, parenchymal and vascular fibrogenic cells. Peribiliary cells preferentially expressed collagen α1(I), while parenchymal myofibroblasts expressed both αSMA and collagen α1(I). In conclusion, these data demonstrate heterogeneity of gene expression in myofibroblastic cells during active fibrogenesis. These reporter mice provide a useful tool to further characterize fibrogenic cell types and to evaluate antifibrotic drugs. (HEPATOLOGY 2004.)