The present study utilized a 2×2×2 factorial design examining age (old vs. young), follicle size (≥2mm vs. <2mm) and media supplementation (with or without fetal bovine serum [FBS]) to determine factors that might affect in vitro maturation of alpaca oocytes. We hypothesized that oocytes collected from follicles ≥2mm from young alpacas and incubated in maturation media supplemented with FBS would have greater maturation rates than those incubated in any other factorial combination. Oocytes were collected from the ovaries of 11 young alpacas (<10 years old) and 14 old alpacas (>11 years old). Oocytes were classified as morphologically normal oocytes (MNO) and deemed suitable for incubation if ≥3 compact layers of cumulus cells and a homogeneous, evenly granulated cytoplasm were observed. Oocytes from each group of follicle sizes were incubated separately and halves of each group were randomly divided and incubated 24h in chemically defined maturation media with or without 10% FBS. Maturation was defined as the visualization of a polar body at the end of the incubation period. Overall, a greater proportion of MNO were collected from follicles ≥2mm than that obtained from smaller follicles, 55% (136/247) vs. 29.6% (162/547), respectively (P<0.05). A greater proportion of oocytes reached maturation when collected from ≥2mm follicles 36% (49/136) than from <2mm follicles 8% (13/162) (P<0.05). For oocytes obtained from ≥2mm follicles of old alpacas, a greater proportion reached maturation when incubated in media supplemented with FBS than when incubated without FBS; 57.6% (19/33) vs. 18.2% (6/33), respectively (P<0.05).