The genome of all densoviruses (DNVs) so far isolated from mosquitoes or mosquito cell lines consists of a 4-kb single-stranded DNA molecule with a monosense organization (genus Brevidensovirus, subfamily Densovirinae). We previously reported the isolation of a Culex pipiens DNV (CpDNV) that differs significantly from brevidensoviruses by (i) having a approximately 6-kb genome, (ii) lacking sequence homology, and (iii) lacking antigenic cross-reactivity with Brevidensovirus capsid polypeptides. We report here the sequence organization and transcription map of this virus. The cloned genome of CpDNV is 5,759 nucleotides (nt) long, and it possesses an inverted terminal repeat (ITR) of 285 nt and an ambisense organization of its genes. The nonstructural (NS) proteins NS-1, NS-2, and NS-3 are located in the 5' half of one strand and are organized into five open reading frames (ORFs) due to the split of both NS-1 and NS-2 into two ORFs. The ORF encoding capsid polypeptides is located in the 5' half of the complementary strand. The expression of NS proteins is controlled by two promoters, P7 and P17, driving the transcription of a 2.4-kb mRNA encoding NS-3 and of a 1.8-kb mRNA encoding NS-1 and NS-2, respectively. The two NS mRNAs species are spliced off a 53-nt sequence. Capsid proteins are translated from an unspliced 2.3-kb mRNA driven by the P88 promoter. CpDNV thus appears as a new type of mosquito DNV, and based on the overall organization and expression modalities of its genome, it may represent the prototype of a new genus of DNV.