The purpose of this study was to evaluate whether a second cycle of contrast-enhanced voiding urosonography (VUS) with no added contrast medium (CM) can increase the detection rate of vesicoureteral reflux (VUR). One hundred twelve consecutive children with a mean age of 2.9 years with 224 kidney-ureter units (KUU) underwent two cycles of contrast-enhanced harmonic VUS. The first cycle of VUS was performed with 3.5-12.5 ml of suspension 300 mg/ml SH U 508 A and was followed immediately by a second cycle with only saline without adding CM. VUR was detected in 57 KUU from 44 children (39%) at the first cycle of VUS. Eight of the remaining 68 non-refluxing children (12%) demonstrated VUR at the second cycle (P=0.045). Most cases of missed reflux at the first cycle were grade II (75%). However, in two KUU from two children missed reflux was grade III. In one child reflux (grade II) was missed on the second cycle. Comparing the second cycle of VUS with the first cycle, concordant findings regarding the presence or absence as well as the grade of reflux were found in 94% of KUU. A second cycle of contrast-enhanced harmonic VUS with no added CM discloses significantly more cases of VUR at no additional cost for the examination.