Herpesviruses or herpesviral sequences have been identified in various bat species. Here, we report the isolation, cell tropism, and complete genome sequence of a novel betaherpesvirus from the bat Miniopterus schreibersii (MsHV). In primary cell culture, MsHV causes cytopathic effects (CPE) and reaches peak virus production 2 weeks after infection. MsHV was found to infect and replicate less efficiently in a feline kidney cell, CRFK, and failed to replicate in 13 other cell lines tested. Sequencing of the MsHV genome using the 454 system, with a 224-fold coverage, revealed a genome size of 222,870 bp. The genome was extensively analyzed in comparison to those of related viruses. Of the 190 predicted open reading frames (ORFs), 40 were identified as herpesvirus core genes. Among 93 proteins with identifiable homologues in tree shrew herpesvirus (THV), human cytomegalovirus (HCMV), or rat cytomegalovirus (RCMV), most had highest sequence identities with THV counterparts. However, the MsHV genome organization is colinear with that of RCMV rather than that of THV. The following unique features were discovered in the MsHV genome. One predicted protein, B125, is similar to human herpesvirus 6 (HHV-6) U94, a homologue of the parvovirus Rep protein. For the unique ORFs, 7 are predicted to encode major histocompatibility complex (MHC)-related proteins, 2 to encode MHC class I homologues, and 3 to encode MHC class II homologues; 4 encode the homologues of C-type lectin- or natural killer cell lectin-like receptors;, and the products of a unique gene family, the b149 family, of 16 members, have no significant sequence identity with known proteins but exhibit immunoglobulin-like beta-sandwich domains revealed by three-dimensional (3D) structural prediction. To our knowledge, MsHV is the first virus genome known to encode MHC class II homologues.