In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV. © 2011 SGM.