The present study evaluated follicular superstimulatory (FSS) and superovulatory (SOV) responses and in vivo embryo production in lactating Simmental cows treated with FSH starting 1 or 2 days after follicle aspiration (FA). The performance of a lengthened superovulation program, named 6dFSH-P36-hCG60, is described. At random stages of the estrous cycle, cows (n = 52) were subjected to ultrasound-guided transvaginal aspiration of all follicles >= 5 mm. After FA, cows were randomly assigned to one of two groups in which FSH treatments started 1 or 2 days after FA (groups FA-1D and FA-2D, respectively). Cows were superstimulated with a total of 500 mu g pFSH over 6 days on a decreasing dose schedule and were pre-treated with a single dose of 400 IU of eCG 24 h before the start of FSH treatments. Follicular superstimulatory (the mean numbers of follicles >= 8 mm on the day of hCG treatment) and SOV responses (the mean numbers of CL and cows with >= 3 CL at the time of collection) were similar in FA-1D and FA-2D groups. However, when compared to FA-1D group, the number of unfertilized ova tended to decrease (0.4 vs 1.7; P = 0.065) and percentage of fertilized ova tended to increase (95.8% vs 84.6%; P = 0.066) in FA-2D group. Moreover, the mean numbers and percentages of both transferable embryos (8.0 and 77.6% vs 6.4 and 57.7%) and freezable embryos (5.3 and 51.5% vs 3.5 and 31.1%) were numerically higher in FA-2D group than FA-1D group.