Tsukamurella pulmonis is an opportunistic actinomycetal pathogen associated with a variety of rarely diagnosed human infections. In clinical cases of infection, T. pulmonis usually accompanies other bacterial pathogens. Because of these mixed infections, a robust diagnostic assay is important. The bacteria cell surface polysaccharides are considered not only useful targets for diagnostics but also intriguing subjects for analysis of the interactions that regulate the host response in general. Here, the structure of the polysaccharide component of the T. pulmonis cell wall was established. Sugar and methylation analysis and 2D-NMR techniques revealed that its polysaccharide belongs to the class of arabinomannan composed of branched tetrasaccharide repeating units, with addition of linear →6)-α-D-Manp-(1→ mannan. Rabbit polyclonal sera against T. pulmonis and T. paurometabola bacterial cells revealed cross reactivity between their antigens. Tissue samples from mice infected with T. pulmonis revealed liver abscesses and pathologic granules located intracellularly when immunohistochemically stained with monoclonal antibodies raised against T. pulmonis polysaccharide. Ultrastructural studies revealed that these granules contain T. pulmonis cells. These observations indicate that T. pulmonis is a pathogenic species capable of spreading within the organism, presumably through the blood.