Seminal plasma is a rich source of ions (e.g. bicarbonate) and proteins which synergistically activate sperm motility required for cervical mucus penetration. C-type natriuretic peptide (CNP) is a 22-amino acid secreted signalling peptide. CNP is present in high concentrations in bull seminal plasma (364 µM¹) and is associated with the bull sperm membrane². In this study, bull spermatozoa, washed by gradient centrifugation, were incubated (37ºC, 180 mins) with a low (L; 0.45 µM), medium (M; 4.5 µM) or high (H; 45µM) dose of CNP in the presence or absence of bicarbonate (25mM). Motility and membrane characteristics were assessed. Bicarbonate supplementation increased the proportion of sperm with a fast and linear trajectory (determined by multi-variate cluster analysis of CASA variables). CNP dose of “M” and “H” in the presence of bicarbonate and “L” and “H” dose in the absence of bicarbonate showed an immediate increase in the proportion of fast-linear sperm. However, after 90 mins, bicarbonate had a divergent effect on response of sperm to all doses of CNP, showing an increase in the fast-linear population when bicarbonate was present and a decrease in its absence. Both bicarbonate and CNP supplementation reduced the proportion of cells exhibiting high mitochondrial membrane potential (detected as orange only fluorescence with JC-1 staining) and increased the “intermediate” population showing both green (monomers) and orange fluorescence (aggregates). Supplementation of CNP had no effect on viability (assessed by PI) or membrane fluidity (assessed by M540) but did cause a small, but significant (P<0.05), increase in the number of non-viable, acrosome reacted cells at the “H” dose of CNP (10.60% to13.03%). We propose CNP is a novel motility activating factor that binds sperm upon ejaculation to initiate fast-linear motility.  Our current research is focused on identifying the signalling pathways (cyclic GMP and/or AMP) involved in this response.