Alpacas are important livestock animals in the Andean highlands of South America. However, their reproductive efficiency is low under natural conditions. In this study, we analysed the oestradiol-17β (E2) and progesterone (P4) levels in the serum and faecal samples of female alpacas before exposure to males for mating to determine whether E2 and P4 affect male acceptance and pregnancy in alpacas. In Experiment 1, faecal samples were collected from nine female alpacas prior to exposure to males for mating to evaluate the effects of premating faecal hormone levels on mating behaviour. In Experiment 2, four of nine female alpacas, which had not previously accepted mating, were injected intramuscularly with oestradiol benzoate (EB). Mating receptivity after EB administration, serum E2 levels before and after EB administration and pregnancy were then evaluated. In both experiments, P4 and E2 were measured in serum and faecal samples using ELISA kits after extraction with diethyl ether and methanol respectively. In Experiment 1, of 23 contacts with males in the mating test, females accepted males 12 times and rejected them 11 times. The proportion of females rejecting males increased as the premating faecal P4 level increased (p = 0.021) and the proportion of females accepting males increased as the faecal E2 level increased (p = 0.035). In Experiment 2, serum E2 levels were higher 2 h after injection than before EB injection (p <
0.05). Alpacas that refused to mate before injection accepted mating 2 h after EB treatment. All alpacas with faecal E2 levels more than 0.5 ng/mg of dry matter before mating had higher P4 levels a month after mating than those with low E2 levels (p <
0.05) and were pregnant. In conclusion, this study showed that measurement of faecal E2 levels can help in determining the appropriate breeding time and that EB treatment improves reproductive performance in alpacas.