We examined patterns of vertical distribution within an assemblage of seven species of large wandering spiders in a lowland rainforest on the Caribbean slope of Costa Rica. Over 16 months, 22 trees were surveyed regularly at night, up to a height of 43 m, using a rope climbing technique. Local climate and canopy microclimate exhibited only weak seasonal fluctuations. There was a distinct vertical segregation between ground-dwelling and arboreal species. The arboreal species used almost the entire available height range, whereby immature Cupiennius coccineus F.O.P.-Cambridge, 1901 occupied higher parts of the trees than adults. The two most abundant arboreal species differed also in their use of arboreal microhabitats: while Cu. coccineus occurred most frequently on trunk bark and on epiphyte leaves, the smaller Ctenus sp. 4 was more restricted to trunk bark. We detected effects of structural complexity of the host trees but no effects of canopy microclimate or of local climate on vertical distribution of Cu. coccineus, by far the most abundant arboreal species. The differences in vertical distribution both between age classes of Cu. coccineus, and between Cu. coccineus and the smaller Ctenus sp. 4 suggest size-dependent habitat segregation in arboreal species along the vertical axis, which might diminish cannibalism and/or intra-guild predation. Moreover, the wide range of vertical distribution of arboreal spiders suggests that they may connect the understory and canopy food webs.