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Parameters which distinguish phototaxis from random motility in Chlamydomonas reinhardtii have been defined with quantitative assays. The phototactic responses in photosynthetic, mixotrophic, and heterotrophic cultures were highest during exponential growth and declined rapidly as the cultures entered stationary phase. In contrast, random motility was relatively constant throughout growth. Phototaxis and motility also differ in their sensitivity to azide and antimycin A. Both of these drugs inhibited phototaxis within 5 min, but motility was unaffected for at least 30 min. Phototaxis and motility have different ion requirements. Optimum motility was observed in the presence of either Ca++ or Mg++; phototaxis required Ca++ and either K+ or NH4+. Photosynthesis is not required for phototaxis, since phototaxis was not inhibited by dichlorophenyldimethyl urea, and a mutant lacking chlorophyll was phototactic.