Shoot tips and leaves from Lavandula officinalis L. of mature greenhouse-grown plants were inoculated aseptically on callus initiation medium. Callus cultures were subcultured on MS-media supplemented with different growth regulators. The influence of growth regulators (2,4-D, BA, NAA and kinetin) on composition of the volatile compounds has been analyzed individually by gas chromatography (GC) for both types of callus tissues as well as the chemical analysis of in vitro produced plant leaves in comparison with the intact mature plants. Results obtained indicated that application of BA enhanced greatly the growth of shoot-tips derived callus which accounted about two times of that shown in MS-free-hormone medium, while application of kinetin is the most suitable regulating substance for enhanced leaves derived callus growth. Results also indicated that no essential oils were synthesized in shoot-tips and leaves derived callus originated from medium free from any growth regulators, while application of all growth regulators investigated enhanced markedly the biosynthesis of essential oils, which varied greatly at different extents. However, comparison of components, content of the oils and chemical composition between in vitro produced plants with those of leaves from plants growing in their natural habitat revealed that an increase in essential oil concentration accounted by 17% in in vitro plant and there is a profound differences in the composition of essential oils, but no considerable differences was observed in chemical composition.