In the first part of this study, monthly infestation by ticks was evaluated on dogs from December 2000 to November 2002 in the rural area of Taiaçupeba, São Paulo. Adults of Amblyomma aureolatum (Pallas) were found on dogs in all months, with a mean prevalence per month of 46.9 ± 15.7% (range, 25–80%). The mean tick relative abundance per month was 2.4 ± 2.7 ticks (range, 0.5–14 ticks), and the mean tick mean intensity per month was 4.7 ± 4.2 ticks (range, 1.5–23.3 ticks). No A. aureolatum immature ticks were found on dogs. In the second part of this study, we studied the life cycle of A. aureolatum in the laboratory. We tested the suitability of six host species for the immature stages and dogs for the adult stage. Tick developmental periods were observed at different temperatures (23, 25, or 27°C), always with RH >95%, which were satisfactory for all free-living developmental stages of the tick life cycle. Chickens and guinea pigs were the most suitable hosts for larvae and nymphs (recovery rates, 18.4–52.2%). Dogs were highly suitable for adult ticks (all females exposed to them were recovered and laid eggs) but were unsuitable for the immature ticks (recovery rates, 0–10%). Based on published host records for A. aureolatum, our results indicate that dogs and birds are primary hosts for adult and immature stages, respectively, of A. aureolatum in nature. In addition, wild guinea pigs are indicated as another potential primary host for immature ticks.