Myxococcus xanthus BtkB is composed of an N-terminal periplasmic domain and a C-terminal cytoplasmic tyrosine kinase domain. The C-terminal cytoplasmic domain of BtkB was autophosphorylated in the presence of [γ-32P]ATP and MgCl2, and the autophosphorylated BtkB was detected with antiphosphotyrosine antibody, suggesting that BtkB is a bacterial tyrosine (BY) kinase. BY kinases have been demonstrated in the production of extracellular polysaccharide (EPS), antibiotic resistance, stress response, and DNA metabolism. Myxococcus xanthus btkB gene was expressed mainly in the growth phase and early stages of fruiting body development. When cultured in nutrient medium at high temperature (37 °C), btkB mutant showed reduced maximum cell density as compared to the wild type. Under starvation conditions, btkB mutant cells formed fruiting bodies and spores about 24 h later than the wild-type strain. The btkB mutant overproduced yellow pigment during development. Also, btkB mutant showed a decrease in EPS production when compared with the wild-type strain. These results suggested that BtkB may play multiple roles in M. xanthus cells.