Embryonic midbrain can be maintained as free-floating roller tube cultures prior to grafting in experimental Parkinson's disease. We examined the influence of pregrafting culture time and pretreatment with brain-derived neurotrophic factor on graft survival and function. Cultures were prepared from solid pieces of embryonic (E14) rat ventral mesencephalon and maintained 4, 8, or 12 days in vitro with or without brain-derived neurotrophic factor (100 ng/ml) and grafted into the striatum of 6-hydroxydopamine-lesioned rats. Graft survival and function were evaluated by amphetamine-induced rotation behavior, number of tyrosine hydroxylase-immunoreactive neurons, striatal reinnervation, and graft volume. Rats receiving untreated tissue cultured for 4 or 8 days displayed no differences in graft quality, while grafts from 12-day-old cultures contained significantly fewer (P < 0.05) tyrosine hydroxylase-immunoreactive neurons (340 +/- 97, 267 +/- 92, and 62 +/- 19) and displayed a lower survival rate (9.6 +/- 2.7, 7.9 +/- 2.7, and 2.6 +/- 0.8% for 4, 8, and 12 days in vitro, respectively). Only rats grafted with 4- and 8-day-old cultures recovered significantly (P < 0.05) from lesion-induced rotations (69.4 +/- 18.6, 70.3 +/- 13.9, and 23.2 +/- 12.1% for 4, 8, and 12 days in vitro, respectively). Striatal reinnervation decreased with increasing culture time (P < 0.05). Pretreatment of the cultures with brain-derived neurotrophic factor affected only graft-induced fiber reinnervation, which was reduced even after short culture times. We therefore suggest that a storage period of 8 days is well suited to maintain embryonic rat ventral mesencephalon with the free-floating roller tube culture technique prior to transplantation. BDNF pretreatment as a new strategy to improve graft survival and function, however, was not effective.