Radionuclide procedures frequently are performed as part of the diagnostic workup of osteomyelitis. Bone scintigraphy accurately diagnoses osteomyelitis in bones not affected by underlying conditions. Degenerative joint disease, fracture, and orthopedic hardware decrease the specificity of the bone scan, making it less useful in these situations. Gallium-67 scintigraphy was often used as an adjunct to bone scintigraphy for diagnosing osteomyelitis. However, now it is used primarily for spinal infections when (18)F-FDG imaging cannot be performed. Except for the spine, in vitro-labeled leukocyte imaging is the nuclear medicine test of choice for diagnosing complicating osteomyelitis. Leukocytes accumulate in bone marrow as well as in infection. Performing complementary bone marrow imaging with (99m)Tc-sulfur colloid facilitates the differentiation between osteomyelitis and normal marrow and improves test overall accuracy. Antigranulocyte antibodies and antibody fragments, such as (99m)Tc-besilesomab and (99m)Tc-sulesomab, were developed to eliminate the disadvantages associated with in vitro-labeled leukocytes. These agents, however, have their own shortcomings and are not widely available. As biotin is used as a growth factor by certain bacteria, (111)In-biotin is useful to diagnose spinal infections. Radiolabeled synthetic fragments of ubiquicidin, a naturally occurring human antimicrobial peptide that targets bacteria, can differentiate infection from sterile inflammation and may be useful to monitor response to treatment. (18)F-FDG is extremely useful in the diagnostic workup of osteomyelitis. Sensitivity in excess of 95% and specificity ranging from 75%-99% have been reported. (18)F-FDG is the radionuclide test of choice for spinal infection. The test is sensitive, with a high negative predictive value, and reliably differentiates degenerative from infectious vertebral body end-plate abnormalities. Data on the accuracy of (18)F-FDG for diagnosing diabetic pedal osteomyelitis are contradictory, and its role for this indication remains to be determined. Initial investigations suggested that (18)F-FDG accurately diagnoses prosthetic joint infection; more recent data indicate that it cannot differentiate infection from other causes of prosthetic failure. Preliminary data on the PET agents gallium-68 and iodine-124 fialuridine indicate that these agents may have a role in diagnosing osteomyelitis.