Objective: Comparison of two frequencies of follicular aspiration in vivo (OPU) in Fleckvieh heifers under tropical conditions, taking as variable responses rates of maturation, cleavage and production of blastocysts in vitro. Material and Methods: Sixteen heifers were used, eight aspirated at once a week intervals (T1); and eight at twice-weekly intervals (T2); two sessions of aspiration per animal, 16 sessions per treatment. An analysis of proportions comparison (Chi square) was carried out in order to analyze the variables and the student's t test was used at significance level P <0.05, considering as main effect the frequency of aspiration. Results: T2 showed a blastocyst production rate (58.82%), significantly higher (P <0.05), than T1 (41.17%). Conclusions: The frequency of aspiration twice a week applied in Fleckvieh heifers under conditions of tropical climate, allows to obtain higher rates of blastocysts production and therefore, better results in a continuous program of In Vitro Embryo Production (PIVE).