The routine determination of sperm concentration not only provides a measure of semen quality but also supplies a means of delivering precise sperm numbers in modern artificial insemination (AI) programs. Methods for estimating sperm numbers in laboratory settings (e.g., hemacytometer) are not practical for use in breeding facilities. A microprocessor-controlled semen analyzer (Densimeter, Model 534-B-Mod1, Animal Reproductive Systems) is commercially available for evaluating stallion semen. We calibrated and modified the semen analyzer to determine sperm concentration in turkey semen and to provide information on the required dilution for a constant AI dose. To calibrate the semen analyzer for turkeys, six samples of pooled semen (10 toms per sample) were collected by abdominal massage. A dilution curve of undiluted and 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:5, and 1:10 diluted semen was established using Beltsville Poultry Semen Extender. Sperm concentration in diluted samples was determined by hemacytometer counts. The hemacytometer counts were used to develop an equation for estimating sperm concentration from the semen analyzer's absorbance readings and the equation programmed into the instrument. The newly developed program on the semen analyzer was subsequently validated by comparing instrument values for sperm concentrations with concurrent hemacytometer counts, which compared well to concentrations determined by a previously calibrated photoelectric colorimeter (Klett). Correlation coefficients between the semen analyzer and hemacytometer counts and the Klett and hemacytometer counts were r = 0.996 and r = 0.992, respectively (P < 0.001). The semen analyzer method was accurate and precise and could be beneficial to commercial turkey AI programs because it is easy to use, requires limited technological skills, and provides results for determining sperm concentration and AI dose in approximately 1 min.