We describe an image analysis (IA) program that has been developed for the quantitative evaluation of mRNA evidenced by in situ hybridization (ISH) with radiolabeled probes in cultured cells and tissue sections. ISH-IA allowed the detection and quantitative evaluation of mRNA expressed by heterogeneous in vitro cultured cells. This method, when combined with dot-blot analysis, allowed the evaluation of the approximate number of mRNA molecules expressed by single cells. IA permitted the evaluation of cultured cells' morphological parameters (such as cytoplasm and nucleus areas) modifications in relation to specific mRNAs expression, which can vary during cell cycle, development, aging, and in different pathologies and treatment with drugs. ISH-IA was applied for the evaluation of mRNA isoforms generated by alternative splicing in single cells. This methodology was also applied for the semiquantitative evaluation and comparison of mRNA levels expressed by different cell types in human normal and tumor tissue sections.