The need for the increased production of honey bee (Apis mellifera) queens has led beekeepers to use different substrates in artificial queen cups where larvae destined to become queens are deposited (grafting). However, not enough scientific evidence exists that indicates that this practice is useful and what substance offers the best results. This study was conducted to determine with the Doolittle queen rearing method the acceptance rate of larvae deposited on different substrates during grafting and to determine if the sugar content and pH of the substrates used affect the acceptance of larvae in cell builder colonies. The evaluated substrates were coconut water, apple nectar, royal jelly, cola soda and distilled water, plus control (without substrate). Grafted larvae of the six treatments were introduced into cell builder colonies and their acceptance verified after 72 h. Apple nectar provided the highest rate of larvae acceptance with 81.06%, followed by cola soda with 62.93%, coconut water with 60.90%, royal jelly with 57.82% and distilled water with 58.99%. The larvae acceptance rates of all substrates were significantly higher than the control, which had an acceptance rate of 47.04%. No significant relationship was found between the sugar content of the substrates and larvae acceptance. However, although not significant, a high negative correlation was found between the substrate pH and the number of accepted larvae (Rho = - 0.90, p = 0.07). These results suggest that the use of liquid acidic substrates during larvae grafting, in particular apple nectar, may increase the production of honey bee queens.