The downstream effects of p15 and p16 gene deletions and loss of transcripts on dihydrofolate reductase (DHFR) were examined in 63 B-precursor (BP) acute lymphoblastic leukaemia (ALL) samples. p15 and/or p16 gene deletions were seen in 6% and 8%, respectively, of BP-ALL samples; however, losses of p15 and/or p16 transcripts were seen in 26 out of 63 (41%) samples. Loss of p15 transcripts (36.5%) exceeded that for p16 (17.5%). For the 26 BP-ALLs that lacked p15 and/or p16 transcripts, only six (23%) exhibited low levels of DHFR by flow cytometry assay with Pt430, a fluorescent anti-folate. Conversely, 18 out of 37 (49%) BP-ALL samples with intact p15 and/or p16 genes and transcripts showed low levels of DHFR (P = 0.04). In p15- and p16-null K562 cells transfected with a tetracycline-inducible p15 cDNA construct, induction of p15 transcripts and protein was accompanied by decreased growth rates, decreased S-phase fraction, decreased retinoblastoma protein phosphorylation, and markedly reduced levels of DHFR transcripts and protein. Collectively, our results suggest that losses of p15 and/or p16 gene expression result in elevated levels of DHFR in BP-ALL in children. However, additional downstream factors undoubtedly also contribute to elevated levels of this enzyme target.