Arginine deiminase (EC 3.5.3.6) from Mycoplasma arthritidis ATCC 14152 has been purified 6-fold by a new procedure, protamine sulfate fractionation and DEAE-agarose chromatography. The yield was 75 to 85%. The homogeneity of the final preparation was demonstrated by gel filtration, sodium dodecyl sulfate-gel electrophoresis, NH2-terminal analysis, and polyacrylamide gel electrophoresis at two pH values. The enzyme has a molecular weight of 80,000 as measured by gel filtration. The dimeric nature of the enzyme is suggested by the molecular weight of 49,000 from sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing in polyacrylamide gels showed a major band corresponding to an isoelectric point of 7.0 and sometimes minor bands having lower isoelectric points. The ultraviolet spectrum exhibits a maximum at 278 nm. The enzyme has high affinity for L-arginine, with a Km value of 4 +/- 1 micronM at pH 7.2, 25 degrees. Mycoplasma arthritidis produces two distinct forms of arginine deiminase. Deiminase I is isolated from cells harvested during logarithmic phase; deiminase II is obtained from late logarithmic or early stationary phase cells. The two forms are resolved by DEAE-agarose chromatography and by polyacrylamide gel electrophoresis. Deiminase II elutes later from a DEAE-agarose column and moves toward the anode faster than deiminase I at pH 9.5 The two forms also have different specific activities and 280:260 spectral ratios. Each form has the same Km and molecular weight. A third form of the enzyme, deiminase III, can be generated by incubating deiminase II at pH 9.8, or in 50% saturated ammonium sulfate, pH 7.0, at 25 degrees. The transformation can be followed by chromatography and is completed within 10 h. The specific activity of deiminase III is 1.3 times that of deiminase II. No change in molecular weight or subunit dissociation was observed during the transformation. Deiminiase III has the same specific activity, absorbance ratio A280:A260, and electrophoretic properties as deiminase I. Deiminase I undergoes no change upon incubation at pH 9.8 for several days.