The aim of this work was to evaluate prospectively the proportion of children with a clinical and biological presentation of acute pyelonephritis, abnormal Tc-99m dimercaptosuccinic acid (DMSA) scintigraphy and negative or equivocal urine cultures. All patients aged 6 weeks to 15 years suspected of acute pyelonephritis (APN) were admitted to the Paediatric Department and underwent Tc-99m DMSA scintigraphy within 3 days after admission and at 6 months. Of 166 patients enrolled in the study, 15 (9%) had negative or equivocal urine culture despite clinical and scintigraphic evidence of APN. Of these 15 children, renal ultrasound was normal in 7 patients and vesicoureteric reflux was found in 9 patients. Control DMSA 6 months after acute episode showed the disappearance of cortical lesions in eight and partial improvement in four patients. In this prospective series, as many as 9% of patients with APN would have been missed on the basis of equivocal or negative urine cultures. It is suggested that Tc-99m DMSA scintigraphy should be performed in children with severe infection without clear aetiology, especially in those with abnormal urinalysis.