There is evidence that calcium influences the migration of neutrophil granulocytes (PMN). As part of the calcium influx pathway, "transient receptor potential" channels (TRP) are involved in the recruitment of PMNs to an infected or inflamed tissue. Here we investigated the role of TRPC1 and TRPC6 in the regulation of intravascular migration of PMNs, which were mechanically stimulated by flow. We used a microfluidic flow chamber to compare neutrophil migration on an endothelial cell layer under static conditions (control) with physiological dynamic conditions (shear stress ~2,040 dyn/cm²). PMNs were isolated from bone marrow of TRPC1^-/- and TRPC6^-/- mice. Migration was analyzed by time-lapse video microscopy. Intracellular Ca²⁺ ([Ca²⁺]_i) was determined with the fluorescent dye fura-2. Migration of TRPC1^-/- and TRPC6^-/- neutrophils was reduced compared to that of wt PMNs. Shear stress reduced velocity and translocation of migrating neutrophils. However, knockout neutrophils were affected more strongly by the onset of flow. After a 10 min flow phase TRPC1^-/- PMNs had acquired a rounded shape and their migration almost stopped. Importantly, knockout neutrophils showed an impaired ability to align with the flow. Surprisingly, following the acute onset of flow, [Ca²⁺]_i rose to a greater extent in both TRPC^-/- than in wt PMNs. This could explain the rounded morphology of knockout PMNs under flow and their reduced migration velocity. In contrast, adhesion under flow was not impaired in the knockout PMNs. Taken together, the lack of TRPC1 & 6 channels severely impairs intravascular migration under shear stress.