We used a flow system to observe the stepwise adhesion and migration of neutrophils on cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumour necrosis factor-alpha (TNF) for 4h, and to evaluate the effects of pentoxifylline (PTX) at each step. When HUVEC had been stimulated with 100 U ml(-1) TNF, treatment of neutrophils with PTX did not reduce the number captured from flow but did cause nearly all adherent cells (> 90%) to roll, whereas most untreated cells became immobilized and similar to 30% transmigrated within minutes. On washout of the PTX, many rolling cells halted and started to migrate. Treatment of the HUVEC with PTX at the same time as 100 U ml(-1) TNF did not affect the number of neutrophils adhering, but there was a significant increase in the percentage of cells rolling even though PTX was no longer present. Thus PTX reduced presentation of activating agents by HUVEC, as well as inhibiting the response by neutrophils to surface-presented activating agent(s). If HUVEC were stimulated with 10 U ml(-1) TNF with PTX, the adhesion of flowing neutrophils was greatly inhibited compared to TNF alone. Surface ELISA indicated that PTX reduced TNF-induced upregulation of E-selectin, This reduction was only sufficient to reduce capture of neutrophils at the low dose of TNF. Thus, by using a flow-based model, we have been able to separate the effects of a multipotent agent such as pentoxifylline, which acts on leucocytes and endothelial cells, at each stage of migration. Copyright (C) 2001 John Wiley & Sons, Ltd.