We reported previously that development of basement membrane (BM) during long-term culture of endothelial cells was associated with increased levels of adhesion and migration of neutrophils when the endothelial cells were stimulated with low concentrations of the inflammatory cytokine, tumour necrosis factor α (TNF). We therefore set out to investigate whether the basement membrane influenced neutrophil behaviour by directly interacting with migrating cells or indirectly modifying the responses of the endothelial cells growing on them. Confluent monolayers of first passage human umbilical vein endothelial cells (HUVEC) were cultured for between 1 and 20 days, and then treated with 0, 1 or 100U/ml of TNF for 4h. The HUVEC were then stripped from the surface and the effects of the BM on cellular behaviour were studied as follows: (i) neutrophils were allowed to settle, adhere and migrate on the BM, with or without prior activation with interleukin-8 (10nM); (ii) fresh HUVEC were seeded and cultured on the stripped BM or on gelatin for 48h, TNF was added for 4h, and then neutrophils were allowed to settle, adhere and migrate on the HUVEC. After 20 days culture, stripped HUVEC left a distinct BM which formed a sheet judged by scanning electron microscopy, and which could be rolled up with a pipette tip. Unstimulated neutrophils did not adhere to BM under any conditions, while activated neutrophils bound in similar numbers regardless of the period of culture or whether the HUVEC had been stimulated or not before stripping. However, the neutrophils migrated more slowly on BM from 20 day cultures than on substrate laid down by day 3 cultures (velocity = 7.9 ± 0.2μm/min vs. 4.6 ± 0.5μm/min; mean ± SEM from 3 experiments; p<0.05 by paired t test). When primary HUVEC were seeded on day 20 BM and stimulated with low dose (1U/ml) TNF, they supported greater adhesion than HUVEC cultured on gelatin (28 ± 9% of added cells adhered vs. 16 ± 4%; mean ± SEM from 4 experiments). Nevertheless, when we measured the migration velocity of transmigrated neutrophils, they were slower under HUVEC grown on BM than under those grown on gelatin (velocity = 7.1 ± 0.2μm/min vs. 10.1 ± 0.5μm/min; mean ± SEM from 4 experiments; p<0.05 by paired t test) These data indicate that substances laid down in the basement membrane directly influence the behaviour of migrating neutrophils. The BM also appears to modify the responses of the endothelial cells growing on it, but culture on day 20 BM does not fully recapitulate the effects of 20 days culture during which BM is deposited.