Nicotiana plumbaginifolia suspension cultured cells were grown on medium supplemented with valine, leucine and isoleucine, singly or in combination. The effects of the three branched-chain amino acids on cell growth rate and on the activity of acetohydroxyacid synthase (AHAS), the first enzyme (and the main regulative site) of their biosynthetic pathway, were studied. Results showed that valine and leucine, at concentrations ranging from 10–4 to 10–3 M, inhibit growth, and at higher doses (from 10–2 to 10–1 M) AHAS activity. Growth, but not AHAS activity, was affected also by isoleucine. The addition of ammonium succinate to the culture medium, in order to counteract a possible general inhibitory effect of these compounds on nitrogen metabolism, relieved only partially their cytotoxicity. Feeding cells with equimolar mixtures of the three amino acids resulted in a minor but reproducible decrease in AHAS level, which was proportional to the dose. A similar result was obtained also on N. plumbaginifolia seedlings, suggesting that in this species a modulation of enzyme level could play a role in controlling the flow of metabolites through the pathway.