14C-salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture. Two SA concentrations (20 mM and 200 mM) were used for comparison. SA was quickly taken up in both treatments, and the 200 mM-treated cells absorbed approximately 15 times that of 20 mMtreated cells within 5 min. More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 mM and 20 mM SA, respectively. SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca 2q to the culture medium in the 200 mM SA treatment. However, EGTA had little or no effect on SA excretion in the 20 mM SA treatment. The data suggest that tobacco suspension-cultured cells may contain both Ca 2q -dependent and Ca 2q -independent pathways for SA excretion. Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 mM but not in the 20 mM SA treatment. These data support the existence of alternative SA excretion pathways in tobacco suspension-cultured cells. Tobacco cells may use both Ca 2q -dependent and Ca 2q -independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 mM, but not at 20 mM.