The occurrence of bacterial infections of the urinary tract in children is exceeded in frequency only by bacterial infections of the respiratory tract. Acute pyelonephritis represents the most severe type of urinary tract infection in children. It not only results in greater morbidity but also has great potential for causing irreversible damage, including renal scarring, hypertension before the age of 30 years and pregnancy complications [1‐3]. Escherichia coli causes most pyelonephritis (PN)—approximately 80‐90% of first infections in children. The pathogenesis of E. coli pyelonephritis is complex, involving the interaction of several factors present in the host and in the invading organism. Host factors predisposing to PN can be anatomic, increased uroepithelial cell adherence, and nonsecretion of P blood group or of the Lewis blood group antigens [4]. Previous investigations have indicated that various virulence factors, such as pili associated with pyelonephritis (pap), afimbrial adhesin I, haemolysin (hly) and cytotoxic necrotizing factor 1 (cnf 1) are useful markers for the detection of uropathogenic E. coli and could therefore be used in the diagnosis of PN [5‐7]. The aim of this study was to determine the role of virulence factors in E. coli urinary isolates from children with pyelonephritis in University Children’s Hospital, Sofia. One hundred and eighty E. coli strains were isolated from the urine of hospitalized children, ages between 5 months and 16 years of age, in the University Children’s Hospital, Sofia. Midstream urine samples were collected after washing the external genitalia with sterile water. One hundred children had clinical acute pyelonephritis, with symptoms: fever, temperature 38.5 °C, flank pain, frequent chills, dysuria, frequency of urination, positive urine culture (10 5 bacteria/mL urine), pyuria (10 white blood cells/high power field), elevated erythrocyte sedimentation rate (20 mm/h), an increased concentration of C-reactive protein (20 mg/L) and reduced concentrating capacity. The clinical diagnosis of cystitis and urethritis was based on a history of frequency and urgency of urination, dysuria, suprapubic pain, and significant bacteriuria with the absence of fever, chills, and flank pain, and normal renal function. The control group comprised of 80 sex- and age-matched healthy children who had sterile urine cultures. Their E. coli strains were isolated from normal intestinal flora. Identification of E. coli was by standard bacteriological