Cyclotron production of 99m Tc is a promising route to supply 99m Tc radiopharmaceuticals. Higher 99m Tc yields can be obtained with medium-energy cyclotrons in comparison to those dedicated to PET isotope production. To take advantage of this capability, evaluation of the radioisotopic purity of 99m Tc produced at medium energy (20)(21)(22)(23)(24) and its impact on image quality and dosimetry was required. Methods: Thick 100 Mo (99.03% and 99.815%) targets were irradiated with incident energies of 20, 22, and 24 MeV for 2 or 6 h. The targets were processed to recover an effective thickness corresponding to approximately 5-MeV energy loss, and the resulting sodium pertechnetate 99m Tc was assayed for chemical, radiochemical, and radionuclidic purity. Radioisotopic content in final formulation was quantified using g-ray spectrometry. The internal radiation dose for 99m Tc-pertechnetate was calculated on the basis of experimentally measured values and biokinetic data in humans. Planar and SPECT imaging were performed using thin capillary and water-filled Jaszczak phantoms. Results: Extracted sodium pertechnetate 99m Tc met all provisional quality standards. The formulated solution for injection had a pH of 5.0−5.5, contained greater than 98% of radioactivity in the form of pertechnetate ion, and was stable for at least 24 h after formulation. Radioisotopic purity of 99m Tc produced with 99.03% enriched 100 Mo was greater than 99.0% decay corrected to the end of bombardment (EOB). The radioisotopic purity of 99m Tc produced with 99.815% enriched 100 Mo was 99.98% or greater (decay corrected to the EOB). The estimated dose increase relative to 99m Tc without any radionuclidic impurities was below 10% for sodium pertechnetate 99m Tc produced from 99.03% 100 Mo if injected up to 6 h after the EOB. For 99.815% 100 Mo, the increase in effective dose was less than 2% at 6 h after the EOB and less than 4% at 15 h after the EOB when the target was irradiated at an incident energy of 24 MeV. Image spatial resolution and contrast with cyclotron-produced 99m Tc were equivalent to those obtained with 99m Tc eluted from a conventional generator. Conclusion: Clinical-grade sodium pertechnetate 99m Tc was produced with a cyclotron at medium energies. Quality control procedures and release specifications were drafted as part of a clinical trial application that received approval from Health Canada. The results of this work are intended to contribute to establishing a regulatory framework for using cyclotron-produced 99m Tc in routine clinical practice. The radioisotope 99m Tc remains indispensable in nuclear imaging. 99m Tc is usually obtained from generators containing the mother isotope, 99 Mo, which in turn is made from highly enriched 235 U ($20%, typically 93%) in nuclear reactors. 99m Tc is eluted in the form of sodium pertechnetate and can be used as is or as the starting material for other 99m Tc radiopharmaceuticals used in a variety of diagnostic applications. Cyclotron production of 99m Tc could be a viable alternativ...