Obtaining normal cells has become increasingly important for use in comparative genetic analytical techniques to examine alterations in gene expression during transformation and progression into malignancy. Normal mesothelial cells are not currently available in cell banks and are essential for comparison of genetic expression analysis in current mouse mesothelioma models. The purpose of this investigation was to extract normal mouse peritoneal mesothelial cells using minimal culture techniques to obtain sufficient cells for gene expression analysis. Mesothelial cells were collected from the mouse peritoneal cavity in vivo with minimal contamination of lymphocytes and macrophages. The cells were cultured for approximately eight days until they were just confluent and retained normal mesothelial phenotype and cytokeratin immunoperoxidase staining.