Variants of the human K562 were developed against the nucleoside analogues cytosine arabinoside, 2 chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross-resistance to all nucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of the different nucleoside analogues in all of the variants. There was a strong overexpression of 5'nucleotidase, measured by rt-PCR and enzyme activity, in all resistant variants. There was a two fold increase of ribonucleotide reductase in the fludarabine resistant line and increased expression of purine nucleoside phosphorylase in the 2 chlorodeoxyadenosine selected line. Karyotypic analysis revealed the loss of a 6(q16;q22) deletion present in the parental line in all of the resistant lines. This portion of chromosome 6 has been shown to contain the gene for 5'nucleotidase. Early events in the transport and metabolism appear to be involved in the resistance mechanisms to nucleoside analogues and are responsible for broad cross resistance to this family of compounds.