The study carried out in plant tissue culture laboratory at Department of Biology - College of Science - University of Al-Qadisiyah, included several experiments on the effect of different growth regulators concentration of auxins and cytokinins on the growth and characteristics of callus of Rosmarinus officinalis L. growing under light and dark conditions. Explant ( tops and the lateral buds with a length of 0.5-1.0 cm) sterilized with ethanol solution and sodium hypochlorate, then growing on MS medium with added different concentrations of growth regulators and incubated in the growth chamber under the influence of lighting factor (light and dark) at a temperature of 25 °C, the data were taken every week and after 45 days of cultivation that. The best combination 2,4-D 1.0 mg/l with BA 0.5 mg/l that gave the largest callus weight in the light 3.75 gm, while in the dark, the best combination 1.0 mg/l NAA and 0.5 mg/l BA gave the largest callus weight 3.60 gm, same combination which gave the highest percentage of callus emergence after 2 weeks was 100% in the dark and 95% in the light. The texture, color and shape of the callus varied as the combination