A herpesvirus was isolated during 2 occurrences of mass mortality among adult catfish Ictalurus melas raised in different farms in northern Italy. The agent replicated in the channel catfish ovary (CCO) cell line from channel catfish I. punctatus, inducing a cytopathic effect similar to that caused by Ictalurid herpesvirus 1 (also referred to as channel catfish herpesvirus, CCV). The new herpesvirus, designated I. melas herpesvirus (IcmHV) did not react with polyclonal rabbit or monoclonal antibodies directed to CCV in either neutralization or indirect immunofluorescence assays. The virions of IcmHV possessed a hexagonal nucleocapsid of 107 nm in diameter surrounded by an envelope with a diameter of 227 nm (n = 20) typical for members of the family Herpesviridae. Virions of IcmHV purified from infected CCO cells contained 17 polypeptides ranging in size from 17.5 to 175 kDa and most differed in molecular weight from those found for CCV. The IcmHV was also distinct from CCV when compared by restriction fragment length polymorphisms (RFLP) of genomic DNA following digestions with the endonucleases Kpn I and Sac I. Lastly, the virulence of IcmHV for channel catfish fry and juveniles, respectively, was demonstrated by experimental infections induced by bath exposure or intraperitoneal injection that resulted in 78 to 96% cumulative mortality in groups of exposed fish. Preventing the introduction of this agent into geographic regions where significant channel catfish production occurs should be a high priority.