The life cycles, under experimental conditions, of many neotropical ticks including Amblyomma brasiliense Aragão have been elucidated, however relatively little information is available with reference to their life cycles under natural conditions. The present study sought to investigate the natural life cycle of A. brasiliense, an aggressive human biting tick, within the Itatiaia National Park (INP), Rio de Janeiro state, Brazil. A total of 245 ticks, collected from wild birds, peccaries (Tayassu pecari) and humans or recovered free-living on forest trails, were included in the analyses. All ticks were identified as A. brasiliense using a combination of morphological and molecular techniques (polymerase chain reaction and nucleotide sequencing). The parasitological data produced in this investigation, were combined with findings from previous studies to elaborate a proposed, natural life cycle for A. brasiliense in the INP, a preserved Atlantic rainforest habitat. It is suggested that, T. pecari serve as primary hosts, with wild birds and small mammals functioning as alternative hosts for immature stages and that humans may act as occasional hosts, possibly in consequence of eco-tourism activities. The molecular analyses performed in this study resulted in the identification and deposition of new sequences for fragments of the mitochondrial genes encoding 12S ribosomal RNA (12S rRNA) and cytochrome c oxidase subunit I (COI). The availability of those sequences will be beneficial to other researchers conducting future or retrospective analysis of tick sequencing data. Finally, in common with previous surveys, molecular screening by PCR for the 17kDa and gltA targets provided no evidence for A. brasiliense as a vector of Rickettsia sp.