The group B entomopoxvirus (EPV) from Amsacta moorei (AmEPV) productively infects only insect cells. A series of AmEPV-lacZ recombinants was constructed in which the lacZ gene was regulated by either late (the AmEPV spheroidin or the cowpox virus A-type inclusion [ATI]) or early (the AmEPV esp [early strong promoter; derived from a 42-kDa AmEPV protein] or the Melolontha melolontha EPV fusolin, fus) virus promoters. When the AmEPV recombinants were used to infect vertebrate cells, beta-galactosidase expression occurred (in >30% of the cells) when lacZ was regulated by either the fus or esp early promoters but not when lacZ was regulated by the late promoters (spheroidin or ATI). Therefore, AmEPV enters vertebrate cells and undergoes at least a partial uncoating and early, but not late, viral genes are expressed. Neither viral DNA synthesis nor cytopathic effects were observed under any infection conditions. When an AmEPV recombinant virus containing the Aequorea victoria green fluorescent protein gene (gfp) under the control of the esp promoter was used to infect vertebrate cells at a low multiplicity of infection, single fluorescent cells resulted, which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, suggesting that vertebrate cells survive the infection and continue to grow. Therefore, AmEPV may prove to be a highly efficient, nontoxic method of gene delivery into vertebrate cells for transient gene expression.