Successful cryopreservation of sperm requires: 1) selection of proper diluent; 2) selection of the best cryoprotectant; 3) determination of freezing and thawing rates for optimum retention of fertilization potential; and 4) removal of any materials deleterious to fertility (e.g., glycerol) before insemination. An economically useful process must allow recovery of sperm with sufficient fertilization capacity to enable maximum use of any given superior male. A series of experiments tested a novel semen freezing container (BioPore CryoCell container) having physical characteristics that permit reproducible freezing and thawing plus facile removal of glycerol from the sample after processing. Experiments tested the effect of: a) residual glycerol; b) initial glycerol concentrations on retention of fertility when samples were frozen and thawed at 6 C/min; c) Beltsville Poultry Semen Extender and Minnesota A buffers used during the dialysis procedure; and d) dialysis time. Respectively, the results were: a) .8% (vol/vol) reduced fertility by 5 to 10%; b) 12% glycerol was superior to 10% and 8% glycerol; c) no difference was observed between the two buffers; and d) 90 and 120 min were both superior to 60 min. Numerous pools of rooster sperm cryopreserved in CryoCell containers and dialyzed after thawing in a prototype BioStore environmental control chamber for 90 or 120 min resulted in a mean fertility of 55.6%. This mean fertility of frozen-thawed sperm was based on 3,263 eggs laid by 400 hens on Days +1 through 9 after inseminations on Days -1, 2, and 5. It is likely that broiler stocks might have lower fertility than that obtained from the Barred Plymouth Rock males and the Single Comb White Leghorn females used in these studies. Nevertheless, the procedure described is the first to consistently result in > 50% fertilized eggs as a result of conventional intravaginal insemination (< 200 x 10(6) sperm in 100 microL extender) of sperm processed after thawing by a procedure amenable to the scaleup required for commercial applications.