Short-term exposure to exogenous RLN2 increases cell motility dose-dependently. Relaxin-induced changes in MDA-MB-231 cellular motility were determined after 24-hour exposure using an 8 μm porous membrane migration assay. Cells which migrated through the 8 μm pores to the underside of the membrane were stained, five microscopic fields were counted for each filter and the results of three independent experiments are shown in the graph as mean ± SEM for (a) MDA-MB-231 after incubation with 100 ng/ml and 500 ng/ml recombinant human (rh) RLN2 and (b) after exposure to secreted RLN2 from stable transfectants seeded in the lower chamber, shown here for MDA/EGFP and MDA/RLN2 clone 23. To assess proliferation, (c) MDA-MB-231 cells were treated with the supernatant of MDA/EGFP control transfectants (SN-EGFP) and of MDA/RLN2 transfectants (SN-RLN2) or with rhRLN2 at 100 ng/ml for 24 hours and proliferation was determined with a bromodeoxyuridine (BrdU) assay. rhRLN2 and secreted RLN2 slightly, but significantly reduced cell proliferation in MDA-MB-231 cells. Experiments were performed in triplicates and data were presented as mean ± SEM. Statistical significance was assessed by (a) one-way ANOVA and with (b, c) student t-test using the SPSS software package (SPSS GmbH, München, Germany). *p < 0.05; **p ≤ 0.005.