HL60 myeloid leukemia cells are extensively used as a differentiation model. We investigated a variant of HL60 which is resistant to differentiation induction (HL60-R) by standard differentiation inducers such as retinoic acid and dimethylsulfoxide (DMSO). To find an explanation for this resistance, we examined nucleotide (NTP) and deoxynucleotide (dNTP) pools in HL60-R and its parent cell line, sensitive to differentiation, HL60-S. We also explored whether these differences led to a difference in sensitivity to various antimetabolites. Drug sensitivity was measured with the tetrazolium (MTT) assay, while nucleotides were measured with anion-exchange HPLC. HL60-R cells were between 2- and 5-fold resistant to the antimetabolites 5-fluorouracil, Brequinar, hydroxyurea and N-(phosphonacetyl)-L-aspartate (PALA), but more sensitive to aza-2’-deoxycytidine (DAC), cytarabine and thymidine (5- to 10-fold). The NTP pools in both HL60 variants showed a normal pattern with ATP being the highest (2530–2876 pmol/106 cells) and CTP being lowest. However, UTP pools were 2-fold higher in the HL60-S cells (p <.01), while CTP and GTP pools were 30% higher (p <.01) compared to HL60-R cells. For the dNTP pools, larger differences were observed, with dATP (50 pmol/106 cells) being highest in HL60-R cells, but dATP was 4-fold lower in HL60-S cells. In HL-60-R, the triple combination retinoic acid, DMSO and DAC increased all NTPs almost 2-fold in contrast to HL60-S. Uridine increased UTP (1.4-fold), CTP (2-fold) and dCTP (1.4.-fold) pools in both cell lines, but thymidine increased only dTTP pools (4- to 7-fold), with a depletion of dCTP. PALA decreased UTP and CTP in both cell lines, but increased ATP (only in HL60-R). Hydroxyurea decreased dNTP especially in HL60-S cells. In conclusion, the pronounced differences in NTP and dNTP pools between HL60-S and HL60-R possibly play a role in the induction of differentiation and drug sensitivity.