The adenovirus E1B 19K gene plays an essential role in transformation of primary rodent cells in cooperation with E1A and in the inhibition of apoptosis during lytic infection. It has been shown that this E1B 19K protein is not necessary for viral DNA replication in human cell lines, such as HeLa and KB. We reported here that the E1B 19K mutant viruses were unable to replicate efficiently in a monocyte cell line, U937. Viral DNA synthesis and late gene expression were found to be defective in U937 cells infected with E1B 19K mutants compared with wild-type virus. Early viral RNA splicing patterns also differ between wild-type and dl337-infected cells. Furthermore, the defect in viral replication could be complemented by dl312 virus defective in E1A expression 4 days after infection with E1B mutants, suggesting persistence of the E1B mutant genome in the infected cells despite defective onset of the late phase of replication. These results imply that E1B 19K is required for efficient viral DNA replication in U937 cells. Inefficient DNA replication is also found in another monocyte cell line, THP-1.