A nucleopolyhedrovirus (NPV) was isolated from diseased larvae of the eastern tent caterpillar, Malacosoma americanum (Fabricius), collected from the northeastern part of Turkey. Light and electron microscopic observations showed that this virus is a multiple nucleopolyhedrovirus (MNPV). The dimensions of the polyhedral inclusion bodies varied from 1.0 to 2.1 mu m, and the sizes of the virions were also measured to be approximately 194.5 nm. Restriction analysis of the new isolate and ManeNPV-T2 (previously isolated from M. neustria larvae) showed that the new virus is closely related to ManeNPV-T2. Furthermore, sequence and phylogenetic analysis of polh and lef-8 also revealed a high degree of homology to M. neustria NPV. Our results indicate that the new virus is another isolate of ManeNPV that infects M. americanum larvae naturally. Therefore, it was named ManeNPV-T3. The virulence of ManeNPV-T3, which was tested against the third instar of M. americanum and M. neustria larvae under laboratory conditions, indicated that 0.6 x 10(5) occlusion bodies per larvae caused 100% mortality within 10 days. The same dose also showed viral infection in Malacosoma castrensis, Plodia interpunctella, Thaumetopoea pityocampa, and Galleria mellonella larvae causing 65%, 60%, 35%, and 25% mortalities, respectively. The findings indicate that ManeNPV-T3 is a promising microbial agent for the biological control of Malacosoma species.