The aim of this study was to evaluate effects of season and lactation status on embryo production in Holstein donors (n = 51). Data from 195 embryo collections (94 from lactating cows and 101 from non-lactating cows), at a dairy farm located in Descalvado, Sao Paulo, Brazil, in 2005, were analyzed. The superstimulation protocol included two 3-mg norgestomet ear implants (Crestar�; Intervet, Sao Paulo, Brazil) and an injection of 3 mg of estradiol benzoate IM (EB) on random days of the estrous cycle. Four days later, 500 IU FSH IM (Pluset�; Calier, Buenos Aires, Argentina) were divided into 8 decreasing doses given 12 h apart. With the seventh FSH injection, 0.530 mg IM of sodium cloprostenol (PGFα Ciosin�; Coopers-Brazil, Sao Paulo, Brazil) was given, and implants were removed with the eighth FSH injection. Twelve hours later, 250 �g IM of gonadorelin (Fertagyl�; Intervet) was given, followed by two AI 12 and 24 h later. Ova/embryos were recovered 6.5 days after the first insemination. The total number of ova/embryos, IETS grades 1, 2, and 3 (viable) and grade 4 plus unfertilized (non-viable), were analyzed by General Linear Model (GLM). Effects of donor, lactation status (lactating or non-lactating), sire, season (1: January through March, n = 56; 2: April through June, n = 37; 3: July through September, n = 50; 4: October through December, n = 52), and their interactions were included in the model. There was an effect of donor on all outcome variables (P < 0.01). There was an effect of season on the total number of ova/embryos (1: 10.2 � 1.2; 2: 6.6 � 1.0; 3: 10.0 � 1.1; 4: 11.2 � 1.2; P < 0.02) and of viable embryos (1: 3.7 � 0.5; 2: 2.4 � 0.5; 3: 5.9 � 0.9; 4: 4.4 � 0.7; P < 0.001). Lactation status influenced the total number of ova/embryos (lactating: 10.9 � 0.9 vs. non-lactating: 8.7 � 0.7; P < 0.001) and the number of non-viable embryos (lactating: 6.7 � 0.8 vs. non-lactating: 4.4 � 0.5; P < 0.01). However, there was no effect of lactation status on the number of viable embryos (lactating: 4.14 � 0.5 vs. non-lactating: 4.29 � 0.5; P > 0.10). In summary, the number of viable embryos in Holstein cows was influenced by season and donor, but not by lactation status.