The aim of this study was to evaluate the roles of 67Ga-citrate and 99Tcm-methylene diphosphonate (99Tcm-MDP) planar and single photon emission tomographic (SPET) imaging in patients with vertebral osteomyelitis. Thirty patients (22 females, 8 males) aged 62.7 +/- 16.4 years (mean +/- s) were enrolled prospectively between May 1995 and May 1998. The patients had been on antibiotics for 7 +/- 4 weeks prior to the study. Histology was available for all but nine patients with mild infections, who were evaluated by a combination of magnetic resonance imaging (MRI), clinical and laboratory tests. 67Ga-citrate (185 MBq) and three-phase bone (555 MBq 99Tcm-MDP) planar and SPET imaging were performed in all patients, together with MRI as a comparison. In total, 67 infectious foci were detected. Based on histology, there were four cases of severe, 13 cases of moderate and four cases of mild osteomyelitis; nine mild infections were also classified by the combination of MRI, clinical and laboratory results. Combined MRI and 67Ga-citrate SPET correctly classified all patients; MRI detected all 67 infectious foci, whereas 67Ga-citrate SPET identified 54 only. False-negative results were seen with all other modalities, especially in cases of mild and moderate infection. 67Ga-citrate SPET identified unsuspected cases of endocarditis (n = 2), paravertebral abscess (n = 1), subaxillary soft tissue abscess (n = 1) and rib osteomyelitis (n = 1). For 67Ga-citrate SPET, the target-to-background ratio was 2.24 +/- 0.31, 1.76 +/- 0.07 and 1.30 +/- 0.18 for severe, moderate and mild osteomyelitis, respectively. Significant differences were noted between severe and moderate infection (P = 0.0051) and between severe and mild infection (P < 0.0001); that between moderate and mild infection was non-significant. For 99Tcm-MDP planar and SPET imaging, and for planar 67Ga-citrate imaging, there was no correlation with severity. We conclude that 67Ga-citrate SPET is able to identify vertebral osteomyelitis and detect additional sites of infection. It can also aid in determining the severity of infection and, potentially, the response to therapy.