Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media. Fundamental knowledge of hairy root physiology is essential to develop and control its culture. In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data logging provides an efficient tool to study rapid physiological variations in hairy root cultures. Datura innoxia hairy roots were grown in a bioreactor equipped with on-line data analyses of pH, dissolved oxygen (pO2), conductivity, oxygen, and carbon dioxide. The experiments were done at a constant temperature and in the absence of light cues. The results obtained showed that the carbon dioxide evolution rate (CER) presented regular oscillations during the culture. Similar oscillations were also observed for the oxygen uptake rate (OUR). These signals were treated mathematically to look for the existence of a rhythm. An autocorrelation function was used to detect any periodic components. The results demonstrate that hairy root respiration exhibited peaks of 1 day. These oscillations, having a period of about 24 h, were also observed in pH and conductivity signals, although not for the pO2 signal. The data acquired in the absence of hairy roots showed that the observed periodic behavior was not an artifact. No effect on rhythms was observed by the imposition of an external "day/night" cycle. The fact that oscillations persisted in the absence of external stimuli, with a free-running period of 24 h, suggests that a circadian rhythm exists in hairy roots of D. innoxia.