Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR IIIa, 55000; TSR IIIb, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, L-cysteine, and β-mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR IIIa, 8.5; and for TSR IIIb, 9.5. The apparent-Km data for DTE and thiosulfate were determined to: [Formula: see text] TSR I, 0.164 mmol·l(-1) and TSR II, 0.156 mmol·l(-1); KmDTE TSR I, 1.54 mmol·l(-1) and TSR II 1.54 mmol·l(-1). The thiosulfate reductases IIIa and IIIb were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.