The extent of multidrug-resistance of human KB carcinoma cell lines has been shown to be proportional to the level of expression of the MDR1 gene. Using an in situ hybridization analysis with 35S-labeled RNA probes, we have found that there is some heterogeneity in expression of the MDR1 gene from cell to cell, but that the average level of expression is proportional to the resistance of the cell line. In the absence of selective pressure, a colchicine-selected multidrug-resistant population with a highly amplified MDR1 gene loses its resistance in parallel with the loss of the amplified gene. Loss of resistance also parallels a decrease in MDR1 RNA expression in the whole cell population. Loss of MDR1 expression in this population is highly heterogeneous, with small clusters of cells maintaining expression even after the population as a whole has become relatively sensitive. This heterogenous loss of expression of the MDR1 gene is consistent with random segregation of amplified DNA segments in the selected cells. The analysis of MDR1 RNA expression by in situ hybridization which is validated by this study should be useful in the study of normal human tissue and tumor samples expressing the MDR1 gene.