We used microarray analysis with Affymetrix rat chips to determine gene expression profiles of freshly isolated rat type I (TI) and TII cells and cultured TII cells. Our goals were 1) to describe molecular phenotypic “fingerprints” of TI and TII cells, 2) to gain insight into possible functional differences between the two cell types through differentially expressed genes, 3) to identify genes that might indicate potential functions of TI cells, since so little is known about this cell type, and 4) to ascertain the similarities and differences in gene expression between cultured TII cells and freshly isolated TI cells. For these experiments, we used preparations of isolated TI and TII cells that contained <2% cross-contamination. With a false discovery rate of 1%, 601 genes demonstrated over twofold different expression between TI and TII cells. Those genes with very high levels of differential expression may be useful as markers of cell phenotype and in generating novel hypotheses about functions of TI and TII cells. We found similar numbers of differentially expressed genes between freshly isolated TI or TII cells and cultured TII cells (698, 637 genes) and freshly isolated TI and TII cells (601 genes). Tests of sameness/difference including cluster dendrograms and log/log identity plots indicated major differences between the phenotypes of freshly isolated TI cell and cultured type II cell populations. The latter results suggest that experiments with TII cells cultured under these conditions should be interpreted with caution with respect to biological relevance to TI or TII cells.