Aim: 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 °C (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%). Conclusion: 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. © 2005, Asian Journal of Andrology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All rights reserved.