Androgenic low-responsive and non-responsive lines obtained as a result of the selection within the hybrid population of Capsicum frutescens L. × C. annuum L. were used for the determination of the pretreatment effect of donor plant with 2,4-dichlorophenoxyacetic acid (2,4-D) on the anther culture effectiveness. A significant increase in embryogenesis of the explants derived from pretreated plants showed an advantageous effect of 2,4-D application. The number of plantlets obtained was 1 to 18 for control and treated plant, respectively. About one half of the plants produced in the anther culture were diploids, characterized by 2C nuclear DNA content (1C/2C – 9/10). Callus tissues were mixoploid and comprised cells from 1C to 8C or from 2C to 16C of the nuclear DNA content. The microspores were the source of the first group of the callus tissue mentioned above. Spontaneous diploidization could be the reason for diploid plantlets regeneration from microspores. Obtaining regenerants in anther culture of a non-responsive hot line, in the case when donor plants were treated with 2,4-D, is the most interesting result of the experiment.