The aim of the present study was to investigate the influence of semen handling by different inseminators on quality of frozen-thawed bovine semen. For this study a total of 10 ejaculates were collected from 7 bulls and semen stored frozen for 7 months in vessels of 10 inseminators each, in three different regions of Switzerland (A, B and C). After storage motility and acrosome integrity (live spermatozoa with intact acrosomes) were measured in frozen-thawed semen by computer-assisted sperm analysis (CASA) and flowcytometry, respectively. In addition, the 56 days non-return rate (NRR) was determined for each inseminator and the relation between NRR of single inseminators and semen quality examined. Results demonstrate that motility as well as acrosome integrity of stored frozen semen were significantly influenced by the specific delivery system used in the different regions and by the ejaculate. After storage of straws in region B semen motility was significantly (P < 0.05) lower than in region C. In region B the percentage live and acrosome intact spermatozoa as well as the NRR of the inseminators were significantly (P < 0.05) lower than in region A. Motility and acrosome integrity of semen stored by single inseminators were significantly correlated with the inseminator's NRR. We conclude that the quality of frozen semen clearly varied between inseminators of the three regions with an impact on NRR.