Clonal cell lines representing different developmental stages of the metanephric mesenchyme were made from transgenic mice with the Simian Virus 40 T-antigen (SV40 Tag) gene driven by the Hoxa 11 promoter. The resulting mK3 cell line represented early metanephric mesenchyme, prior to induction by the ureteric bud. These cells showed a spindle-shaped, fibroblast morphology. They expressed genes characteristic of early mesenchyme, including Hoxa 11, Hoxd 11, collagen I, and vimentin. Moreover, the mK3 cells displayed early metanephric mesenchyme biological function. In organ co-culture experiments they were able to induce growth and branching of the ureteric bud. Another cell line, mK4, represented later, induced metanephric mesenchyme undergoing epithelial conversion. These cells were more polygonal, or epithelial in shape, and expressed genes diagnostic of late mesenchyme, including Pax-2, Pax-8, Wnt-4, Cadherin-6, Collagen IV, and LFB3. To better define the gene expression patterns of kidney metanephric mesenchyme cells at these two stages of development, RNAs from the mK3 and mK4 cells were hybridized to Affymetrix GeneChip probe arrays. Over 4000 expressed genes were identified and thereby implicated in kidney formation. Comparison of the mK3 and mK4 gene expression profiles revealed 121 genes showing greater than a ten-fold difference in expression level. Several are known to be expressed during metanephric mesenchyme differentiation, but most had not been previously associated with this process. In situ hybridizations were used to confirm that selected novel genes were expressed in the developing kidney.