The aerial tissues of Tilia americana var. mexicana produce compounds with anxiolytic activity, such as quercetin 3-O-β-D-glucoside and tiliroside, in addition to ones with anti-inflammatory properties, such as scopoletin. These three compounds were initially identified in callus cultures of apical buds. In the present study, suspension cultures from leaf explant callus were established; the accumulation of scopoletin and quercetin 3-O-β-D-glucoside in these cultures were found to be cell-growth-associated using cell growth and active compound-production kinetics assays. The effects of varying the nitrate and copper concentrations in Murashige and Skoog (MS, 27.4 mM total nitrates and 0.01 µM copper) medium on the growth of a suspension culture of T. americana cells and on the production of active compounds were tested by means of central composite design (CCD) generally used in the response surface methodology (RSM). Cell growth, measured as maximal biomass, improved when the total nitrate concentration decreased in the MS medium to 13.7 mM (p