Neutrophils as part of the innate immune system play a major role in the inflammatory response. Attracted by chemokines they migrate into the affected tissue. Chemotaxis involves binding of chemokines to their receptors leading to the activation of the respective signaling pathways that involve intracellular Ca²⁺ transients. On our way to identify the involved Ca²⁺ entry channels we investigated the role of TRPC6 channels. We studied the impact of TRPC6 knockout on chemotaxis of murine neutrophils in 3-dimensional matrices with time-lapse videomicroscopy. For chemotaxis assays fMLP was used as end-target chemoattractant and as intermediate chemokine an ex-vivo chemokine cocktail was applied. The chemokine cocktail was a product of a mouse peritonitis induced with casein and consisted of the peritoneal exudates that were collected after 5 hours of casein treatment. Chemokine receptor signaling was studied via Western blot. Chemotaxis of TRPC6^-/- neutrophils along the chemokine cocktail gradient was strongly impaired while chemotaxis towards fMLP was not affected. The "motor of the migration" was hardly affected in TRPC6^-/- neutrophils since their migration velocitiy was only slightly reduced. Blocking of chemokine receptor CXCR2 led to a flattened chemotaxis of WT cells but had no effect on TRPC6^-/- cells. Western Blot analysis indicated that CXCR2 expression was not influenced by TRPC6 but signaling of CXCR2 via pAKT or p38 Map kinase was impaired in TRPC6^-/- cells. Our findings indicate that TRPC6 is involved in chemotaxis of murine neutrophils. In this context TRPC6 might be a component of intermediate chemoatractant signaling.