In this study, we evaluated the fish ascent in the experimental ladder at Itaipu Reservoir (Paraná State, Brazil), in relation to diel variation, fish size, reproductive stages and species composition. The study was carried out from October, 1995 to February, 1996 and from November, 1996 to January, 1997 at two points on the ladder (boxes at heights of 10 and 27 m). Twenty–two species were recorded, and Prochilodus lineatus, Pimelodus maculatus, Leporinus obtusidens, Schizodon borellii and Leporinus friderici accounted for 86.6 % of the total catch. Fish length did not differ between diel periods and boxes. On the other hand, fish abundance of the main species was significantly influenced by box position, reproductive stage and diel period. The higher box showed lower abundance, suggesting some selectivity in ascension. The ability to ascend the ladder did not show any relationship with reproductive migration, since non–reproductive adults comprised most of the catch. An interesting finding is that the major taxonomic groups showed specific diel preferences in ascending the ladder: Characiformes ascended the ladder predominantly during daylight, whereas Siluriformes migrated at night. These results emphasize how complex ladders are as management tools, and stress the need to acquire information on several aspects as a condition to achieve conservation goals.