Neutrophils isolated from the peripheral blood of pigs free of infection withMycoplasma hyopneumoniae were loaded with a fluorescent indicator (Fura-2) for detection of cytosolic free calcium concentration. The kinetics of the intracellular calcium flux were examined after incubation with or without a pathogenic or a non-pathogenic strain ofM. hyopneumoniae. The basal intracellular calcium concentration was not altered by incubation withM. hyopneumoniae. However, the relative increase in cytoplasmic calcium concentration caused by the addition of opsonized zymosan was significantly (p<0.05) higher in neutrophils incubated withM. hyopneumoniae as compared to neutrophils not incubated withM. hyopneumoniae. Additionally, after zymosan stimulation, the intracellular calcium concentration was greater in neutrophils incubated with a pathogenic strain ofM. hyopneumoniae than in those incubated with a non-pathogenic strain. This suggests thatM. hyopneumoniae alters the signal transduction mechanisms in neutrophils and that this alteration may be related to virulence.