Didelphids use vertical strata in different ways, suggesting the existence of a wider variety of niches than simply arboreal or terrestrial. This variety could be related to the differential ability to deal with support diameters and inclines, and might be important for the coexistence of species in local assemblages. From data obtained with the spool-and-line technique, the use of the vertical space and above-ground supports is described in four species of an assemblage of didelphid marsupials in south-east Brazil. The stratification in this and other assemblages in the Atlantic forest and in the Amazon is also compared. Animals were trapped in Serra dos Órgãos, state of Rio de Janeiro, and equipped with a spool-and-line device before release. The paths were tracked by measuring variables related to support diameter, incline, distance and height moved above ground. The diameter of supports used was positively related to body size, and the incline to the dominant direction of movement (horizontal vs vertical). The more cursorial species, Metachirus nudicaudatus, only once moved above ground in > 3200 m of paths followed. Didelphis aurita moved mostly on the ground, but was the only species that occasionally reached the canopy. Philander frenata also moved mostly on the ground, occasionally used the understorey, but never reached the canopy. The more arboreal species, Marmosops incanus, moved mostly in the understorey, but never in the canopy. This pattern of stratification is similar to that observed in other sites in the Atlantic forest and in the Amazon.