Emerging evidence indicates a close connection between components mitochondrial electron transport chain (mETC), nitric oxide (NO) and alternative oxidase (AOX) activity in plants. In unicellular algae, AOXs are monomeric fungi-type proteins. We previously showed that in Chlamydomonas reinhardtii stress-induced AOX1 expression was significantly higher under dark conditions. Here we found that in dark aerobic Chlamydomonas cells, complex III of mETC produces NO from nitrite, and monomeric AOX1 appeared to be involved in reducing this activity. Moreover, in dark-incubated cells, NO generation is not mediated by nitrate reductase and nitric oxide-forming nitrite reductase complex. Under conditions limiting the flow of electrons through complex III, the expression of AOX1 but not the AOX2 expression is dependent on changes in NO levels. Taken together, the data indicate that in the absence of light, NO generated by mETC, is a key molecule controlling AOX1 levels in Chlamydomonas aerobic cells.