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Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca fascicularis) spermatozoa; instead, both have an absolute requirement for exogenous stimulation with caffeine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting criteria for automated analysis of macaque sperm exhibiting hyperactivated motility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteristics were assessed by computer-assisted motion analysis. Tyrosine phosphorylation of sperm tail proteins was determined by immunocytochemistry with PY-20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) ≥ 150 μm/sec; amplitude of lateral head displacement (ALH) ≥ 8.0 μm, and linearity (LIN) ≤ 60%. Using these criteria, caffeine and dbcAMP significantly stimulated HA (61 ± 8%) compared to control conditions (12 ± 2%), p < 0.01, with a concomitant increase in PY-20 labeling (88 ± 12%) vs. control (13 ± 2%), p < 0.01. PY-20 labeling significantly correlated with HA (r = 0.75, p < 0.01) and with some motion characteristics used for HA sorting including ALH (r = 0.86, p = 0.0013) and LIN (r = −0.88, p < 0.001) but not VCL (r = 0.21). Treatment with genistein (10 μM) had no effect on HA or PY-20 immunocytochemistry in the absence of caffeine and dbcAMP, but the tyrosine kinase inhibitor significantly decreased caffeine- and dbcAMP-stimulated HA and PY-20 labeling in a dose-dependent manner (p < 0.01). These results demonstrate that tyrosine phosphorylation of sperm tail proteins is an integral signaling pathway modulating some but not all of the motion characteristics associated with cAMP- and caffeine-stimulated HA in cynomolgus monkey spermatozoa.