OBJECTIVE To evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation. METHODS Semen samples were obtained from four adult alpacas (Lama pacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I-G; 2) extender I-EG; 3) extender II-G; and 4) extender II-EG. Semen was diluted in a two-step process: for cooling to 5 degrees (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. RESULTS When compared, the motility after thawing was higher (P >0.05) in groups II-EG (20.0% +/- 6.7%) and II-G (15.3 % +/- 4.1% ) than that in groups I-G (4.0 % +/- 1.1%) and I-EG (1.0 % +/- 1.4%). Viable spermatozoa with intact acrosomes in groups II-EG (18.7 % +/- 2.9%) and II-G (12.7 % +/- 5.9%) were higher than that in groups I-G (5.7% +/- 1.5%) and I-EG (4.0% +/- 1.0%). CONCLUSIONS The skim milk- and egg yolk-based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders.