Abstract: A series of experiments were conducted to quantify PGF2α in bovine semen, seminal plasma, and extended semen, and to determine if PGF2α was synthesized or released during extension of bovine semen. Concentrations of PGF2α were measured in paired samples of whole and extended semen from beef and dairy bulls. Concentrations of PGF2α did not differ between beef and dairy (mean ± SEM, 273.8 ± 42.8 vs. 210.3 ± 18.5 pg/mL, respectively; P = 0.12), but tended (P = 0.08) to be greater for whole compared with extended semen (255.5 ± 29.8 vs. 194.5 ± 17.0 pg/mL). Whole semen was extended at eight dilution rates (regardless of initial sperm concentration), using a diluent consisting of two fractions. Samples collected after each dilution step resulted in four subsamples. Concentrations of PGF2α in subsamples decreased (P < 0.001) at higher dilution rates and later steps of extension. Subsequently, whole semen and seminal plasma were extended at three dilution rates. Initial PGF2α concentration was greater (P < 0.001) for whole semen compared with seminal plasma. During extension, PGF2α synthesis or release resulted in less disparity, but the amount synthesized or released was greater (P = 0.03) for semen compared with seminal plasma. We concluded that synthesis or release of PGF2α during extension resulted in concentrations similar to whole semen. [Copyright &y& Elsevier]