Previous studies have demonstrated the usefulness of flow cytometry in the analysis of dihydrofolate reductase (EC 22.214.171.124) gene amplification. However, this powerful and potentially sensitive method for analyzing gene expression in individual cells has not seen widespread use. This is due in part to the difficulty of producing fluorescent methotrexate (Fluo-MTX), which is needed to label dihydrofolate reductase in vivo, in yields higher than 1% and of sufficient purity to give low nonspecific backgrounds by the published procedures. We have significantly improved the synthesis of Fluo-MTX to obtain rapidly a chromatographically pure product in 20% yields. In addition, we have found that cell volume is a variable which makes direct comparisons of fluorescence intensity between cell lines difficult. In order to circumvent this problem, we have improved flow cytometric analysis to measure the fluorescence specific intensity of individual cells. A survey of various cells commonly used for gene transfer shows a significant variability in the efficiency with which they are labeled with Fluo-MTX, which appears to be due to variations in their ability to transport this reagent.