In order to improve the growth of Zataria multiflora tissues in vitro experiments in different hormonal treatments and culture media were carried out. Shoot cultures, established from plantlets shoot apex, were treated by different levels of 6-benzylaminopurine and 1-naphtalen acetic acid added to Murashige and Skoog medium with two conditions of active charcoal. The highest proliferation rate was obtained with 1 mg L(-1) 6-benzylaminopurine, without charcoal. Callus cultures of Zataria multiflora were established from plantlets shoot nodes on MS supplemented with 1 mg L(-1) kinetin and 0.5 mg L(-1) 2, 4-dichlorophenoxyacetic acid. Callus growth rate on three different media (MS salts and vitamins, MS salts with Gamborg vitamins and Gamborg salts and vitamins) was evaluated. The best growth was recorded on Gamborg medium (salts and vitamins) with 0.75 mg L(-1) 6-benzylaminopurine and this medium was retained for rosmarinic acid production optimization experimentation. Sucrose and glucose were tested at different doses to compare the effect of carbohydrates on the growing rate and the production ofrosmarinic acid in shoot cultures and callus cultures. Experiments also were done at two light exposures (total obscurity or 16 h light/8 h dark). Very high level of rosmarinic acid was detected in callus tissues (158.26 mg g(-1) dry weight) as compared with propagated shoots (12.28 mg g(-1) dry weight). The dose of carbohydrate had a direct effect on the production of rosmarinic acid dependent of the type of tissues. The best ratio production/growth was obtained in callus cultures treated with glucose 75 g L(-1) under light exposure. These results indicate that, in Zataria multiflora, the undifferentiated state of tissues appears particularly more efficient for metabolic production in vitro, such as rosmarinic acid, in comparison to differentiated shoots.