Mycoplasmas (class Mollicutes) are the smallest self-replicating bacteria. These bacteria lack a rigid cell wall and are parasites, exhibiting strict host and tissue specificities (Baseman & Tully, 1997; Rosengarten et al., 2000). Many mycoplasmas are pathogenic to humans and animals and are frequent contaminants of cell cultures (Rottem, 2003). Mycoplasma hyorhinis was first isolated from the respiratory tract of young pigs (Kobisch & Friis, 1996). This organism has been implicated in a variety of diseases in swine (Morita et al., 1995); Kobisch & Friis, 1996) and was shown to be the major contaminant of tissue cultures (Kotani et al., 1990). Interest in M. hyorhinis has been recently further increased after the detection of this organism in human gastric cancer tissues, suggesting a possible association between M. hyorhinis and carcinogenesis (Huang et al., 2001; Yang et al., 2010). A practically noncultivable mycoplasma tentatively identified as M. hyorhinis (to be referred to as strain MCLD) has recently been identified in LB33mel A1, a melanoma cell line. This organism was adapted to grow in a modified mycoplasma medium (Hayflick & Stinebring, 1960; Kornspan et al., 2010). Although M. hyorhinis has been considered to remain attached to the surface of host cells, we have recently shown that MCLD invades nonphagocytic eukaryotic cells (Kornspan et al., 2010). There is no doubt that invasion is associated with the attachment of the organisms to the host cells; nevertheless, attachment is not sufficient to trigger events that lead to invasion (Rosengarten et al., 2000). The invasion of MCLD may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. As phospholipids represent the major chemical constituents of the lipid bilayer, phospholipases are likely to be involved in the membrane disruption process (Weltzien, 1979; Vernon & Bell, 1992). Furthermore, phospholipases may play a fundamental role serving to generate signals required for invasion as well as an array of metabolites with distinct biologic function (Nishizuka, 1992). Cleavage of phospholipids by a mycoplasmal phospholipase C (PLC) will release diacylglycerol that activates protein kinases (Nishizuka, 1992). The activity of phospholipase A (PLA) will release free fatty acids (FFA) as well as lysophospholipids that may perturb the host cell membrane and generate active metabolites (Weltzien, 1979; Vernon & Bell, 1992). Evidence for PLC activity in a variety of mollicutes has been presented before (De Silva & Quinn, 1987; Shibata et al., 1995), and a potent phospholipase A1 (PLA1) was described in Mycoplasma penetrans (Salman & Rottem, 1995). In the present study, we show that M. hyorhinis possess PLA and glycerophosphodiesterase (GPD) activities. The possible role of these enzymes in the virulence of M. hyorhinis and in triggering signal cascades in the host cells is discussed.