SummaryPhenytoin sodium (5–50 μg/ml) caused a dose‐dependent prolongation of the doubling time of the human promyelocytic leukaemia cell line, HL60. This effect was unassociated with any alteration in cell viability. HL60 cells which were pre‐incubated with 15 μg/ml phenytoin sodium for 1 or 48 h and then incubated with the same concentration of the drug plus either3H‐methotrexate (3H‐MTX) or57Co‐cyanocobalamin for 90 min, showed an altered accumulation of both radioactive compounds when compared with control cells. Control cells were not pre‐incubated with the drug and were subsequently studied in the absence of the drug. Pre‐incubation with the drug for 1 h resulted in a 34% increase, and preincubation for 48 h in a 19% reduction in the accumulation of3H‐MTX. Preincubation for 1 or 48 h caused a 29% reduction in the accumulation of57Co‐cyanocobalamin. Cells cultured in the presence of 15 μg/ml phenytoin sodium for 48 h also gave a slightly increased deoxyuridine‐suppressed value; this abnormality was partially corrected by the addition of 50 μg/ml folinic acid to the test system but was unaffected by the addition of 1 μg/ml cyanocobalamin. The data indicate that the effects of phenytoin sodium on the proliferation of HL60 cells may have been slightly mediated via a reduced uptake of folate and possibly also of vitamin B12. They also suggest that one of the mechanisms underlying some of the undesirable effects of long‐term therapy with phenytoin may be a drug‐related impairment of both folate and vitamin B12uptake by certain cells, including