In this study, we investigated the mechanism of the loss or decreased expression of beta 2-microglobulin (beta 2m) in several drug-resistant sublines of MCF-7 and in a doxorubicin (DOX)-resistant variant of the T-47D breast cancer cell line. beta 2m protein and RNA are not expressed in highly metastatic, multidrug-resistant MCF-7/Adr cells with high resistance to DOX. Nuclear run-on transcription and RNA stability assays demonstrate that while beta 2m in MCF-7/Adr cells is transcribed, its mRNA is rapidly degraded after synthesis in these cells, indicating that it is controlled by post-transcriptional mechanisms. We also show that an MCF-7 subline (MCF-7/Adr-5) expressing a very low level of resistance to DOX has a decreased level of beta 2m expression. Treatment with actinomycin D revealed that the half-life of beta 2m mRNA in MCF-7 and MCF-7/Adr-5 cell lines was comparable. Nuclear run-on transcription analysis revealed a decreased rate of beta2m transcription in MCF-7/Adr-5 cells compared to that in MCF-7 cells. Moreover, beta 2m mRNA remained undetectable in MCF-7/Adr cells following cycloheximide treatment. However, in MCF-7 cells, increased beta 2m mRNA was observed after 12 h, and a similar level of increased mRNA expression was observed after 36 h of cycloheximide treatment in MCF-7/Adr-5 cells; these results suggest that one of the mechanisms controlling beta 2m mRNA expression might be a negative regulatory protein in MCF-7/Adr-5 cells. Analysis of the beta 2m status of other drug-resistant MCF-7 sublines revealed that deregulation of beta 2m is not limited to DOX resistance, but can also be detected in cells selected for resistance to mAMSA and DOX-verapamil. In addition, our data show that reduced beta 2m expression correlates with the decreased levels of estrogen receptor (ER) expression in the DOX-resistant MCF-7/Adr and T-47D/Adr-4 human breast cell lines. Furthermore, we provide evidence that the partial inhibition of beta 2m by antisense RNA results in 2-3-fold decreased sensitivity of MCF-7 cells to DOX and mAMSA. Moreover, the addition of exogenous beta 2m protein near its physiological human serum concentration can modulate the DOX sensitivity of the MCF-7 antisense beta 2m and control transfectants. Therefore, these results indicate that lost or decreased beta 2m expression is involved in the development of the drug-resistant phenotype and correlates with the loss of ER in human breast cancer cell lines.