Severe impairment of methotrexate membrane transport in methotrexate-resistant K562 (K500E) cells was characterized by a nearly complete loss of reduced folate carrier (RFC) transcripts and RFC protein. As determined by 5'-rapid amplification of cDNA ends (5'-RACE), approximately 93% of the RFC transcripts in wild-type cells contained the KS43 5'-untranslated region transcribed from the RFC-B promoter. KS43 transcripts decreased > 90% in K500E cells. The basal and full-length RFC-B promoters were more active (3- and 2-fold, respectively) in directing transcription of a luciferase reporter gene in K500E than in wild-type cells. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, or with a histone deacetylase inhibitor, trichostatin A, did not increase the levels of RFC transcripts in K500E cells. No differences in RFC gene structure were detected between the lines on Southern blots; however, the RFC signals were decreased approximately 60% in K500E cells. DNA sequences were identical between the lines for the RFC coding region and the two 5'-non-coding exons and their respective promoters. Spectral karyotype analysis and fluorescence in situ hybridization in wild-type cells showed two normal chromosome 21 copies and one or two marker chromosomes, each with an RFC signal. In K500E cells, the RFC gene locus was no longer localized to a normal chromosome 21 (at 21q22.2), and a single RFC signal was associated with a small metacentric chromosome, characterized by a 21/22 translocation. Our results suggest that loss of RFC transcripts in K500E cells is unrelated to changes in the levels of critical transcription factors, or to differences in the extent of RFC promoter methylation or core histone deacetylation. Rather, this phenotype is due to the loss of one or more RFC alleles, and to a translocation of the remaining RFC allele with the formation of a 21/22 fusion chromosome.