Polymorphonuclear cells (PMNs) attend to inflammatory sites by chemotactic movement, caused by chemoattractants (CAs) like n-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and interleukin-8 (IL-8). However, distinct but applicable assays for investigations of PMNs' migration limit in vitro examination. We integrated CD15-bead-based isolation of PMNs with analysing their chemotaxis in a novel 3D-mu-Slide migration chamber. The PMNs were exposed to different concentrations of FMLP and IL-8 (1, 10 and 100 nM) and observed for 180 min in cell-physiological environment conditions. Moving PMNs' percentage (median and interquartile range) decreased from 62% (27%) to 36% (31%) without CA, from 88% (30%) to 22% (26%) for 1 nM IL-8, from 70% (22%) to 28% (13%) for 100 nM IL-8, from 30% (23%) to 18% (46%) for 1 nM FMLP and from 76% (20%) to 28% (13%) for 100 nM FMLP. Centres of cell movement turned towards the CAs (negative values) within a single 30-min observation period: 5.37 mu m (16.82 mu m) without CA, -181.37 mu m (132.18 mu m) with 10 nM and -239.34 mu m (152.19 mu m) with 100 nM IL-8; -116.2 mu m (69.07 mu m) with 10 nM and -71.59 mu m (98.58 mu m) with 100 nM FMLP. FMLP and IL-8 ensure chemotaxis without increase of chemokinesis. 3D-mu-Slide chemotaxis chambers facilitate time course analyses of PMNs' migration in stable conditions over a long time with concise distinction of chemotaxis and chemokinesis.