Cdc25B protein phosphatase represents an attractive potential therapeutic target for small molecule intervention because of its central role in positively regulating cyclin dependent kinases and thus cell proliferation, as well as its elevated levels observed in many human tumors. Among the most potent previously identified Cdc25 inhibitors have been quinoline quinones, which have a rich legacy as therapeutic agents but have also been associated with nonspecific interactions. In this study, we have interrogated the structure-activity relationship of a focused series of C2-, C3-, or C4-modified quinoline-5,8-quinones on Cdc25B inhibition in vitro. Substitution at the C3-position in this small chemical series were slightly superior to substitutions at the C3-position. For all compounds, recombinant human Cdc25B was approximately 5-fold more sensitive compared to recombinant human PTP1B. Two compounds inhibited HeLa cell growth with IC50 values of approximately 2 microM. Consistent with other para-quinones, some members of this series generated intracellular reactive oxygen species and the in vitro enzyme inhibition was mitigated by addition of reductants or catalase. These results indicate that chemical modifications on the pyridine core are tolerated, providing additional sites for future structural modification of this biologically active pharmacophore.