Objective: To determine the relationship between the virus burden in infancy and hearing loss in congenital CMV infection. Study design: A cohort of 76 infants with congenital cytomegalovirus (CMV) infection identified by means of newborn virologic screening was monitored for outcome. The amount of infectious CMV was analyzed in urine specimens obtained during early infancy. Peripheral blood (PB) samples obtained during early infancy were available from 75 children and CMV DNA was quantitated with a real-time quantitative polymerase chain reaction. Results: Infants with clinical abnormalities at birth (symptomatic congenital CMV infection) had higher amounts of CMV in urine (P =. 005) and CMV DNA in PB (P =. 001) than infants with no symptoms. Eight children with and 4 children without symptoms had hearing loss. Among children without symptoms, those with hearing loss had a significantly greater amount of CMV in urine (P =. 03) and PB virus burden (P =. 02) during infancy than those with normal hearing. Infants with <5 × 103 pfu/mL of urine CMV and infants with <1 × 104 copies/mL of viral DNA in PB were at a lower risk for hearing loss. Conclusion: In children with asymptomatic congenital CMV infection, hearing loss was associated with increased amounts of urine CMV and PB CMV DNA during early infancy. Copyright © 2005 Elsevier Inc. All rights reserved.