PURPOSE: 3-AP (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is a metal chelator that potently inhibits the enzyme ribonucleotide reductase, RR, which plays a key role in cell division and tumor progression. A sub-unit of RR has a non-heme iron and a tyrosine free radical, which are required for the enzymatic reduction of ribonucleotides to deoxyribonucleotides. The objective of the study was to determine whether 3-AP affects its targeted action by measuring EPR signals formed either directly or indirectly from low molecular weight ferric-3-AP chelates. METHODS: Peripheral blood lymphocytes were collected from patients with refractory solid tumors at baseline and at 2, 4.5 and 22 hours after 3-AP administration. EPR spectra were used to identify signals from high-spin Fe-transferrin, high-spin heme and low-spin iron or copper ions. RESULTS: An increase in Fe-transferrin signal was observed, suggesting blockage of Fe uptake. It is hypothesized that formation of reactive oxygen species by FeT(2) or CuT damage transferrin or the transferrin receptor. An increase in heme signal was also observed, which is a probable source of cytochrome c release from the mitochondria and potential apoptosis. In addition, increased levels of Fe and Cu were identified. CONCLUSION: These results, which were consistent with our earlier study validating 3-AP-mediated signals by EPR, provide valuable insights into the in vivo mechanism of action of 3-AP.