ABSTRACT In the recent years, research on the effects of rejuvenation on hot mix asphalt (HMA) with a high amount of RAP (Reclaimed Asphalt Pavement) has intensified. However, the vast majority of scientists focus on the technology of preheated RAP addition (e.g. in the parallel dryer). It was decided to investigate the effect of rejuvenators on asphalt mixtures containing non-preheated RAP (RAP cold addition into the mixer). Different types of rejuvenators were used in the research. The tests were carried out for the binder course and the base course. The optimal amount of cold RAP in the mixtures was determined and based on the initial tests of stiffness modulus and air void content, the amount of rejuvenators was adjusted for later tests. Tests of air void content, stiffness, resistance to water and frost and rutting resistance were carried out in the laboratory. Afterwards tests in the asphalt mixing plant were conducted for the rejuvenator with the best results for the base course. The results obtained confirmed the potential of using rejuvenators for mixtures with cold RAP addition into the mixer. It has been shown that bio-based rejuvenators are suitable for the use in different types of asphalt mixtures.