Quality and in vitro fertilizing ability of frozen-thawed cat semen collected by urethral catheterization (CT) or electroejaculation (EE) after medetomidine administration were compared. Sperm collection was performed by an urinary tomcat catheter and, 4 days apart, by electroejaculation from each of eight tomcats. Results showed that semen collected by CT was characterized by lower volume (10.5 ± 5.3 μL, P < 0.05), higher sperm concentration (1868.4 ± 999.8 × 10 6/mL, P < 0.05) and lower pH (7.0 ± 0.4, P < 0.05) than that collected by EE (67.1 ± 25.9 μL, 542.9 ± 577.9 × 10 6/mL, and 7.9 ± 0.4, respectively). Spermatozoa characteristics after thawing at 0, 3 and 6 h did not differ between the two methods of collection. Also cleavage rate and embryo production from oocytes fertilized with frozen-thawed spermatozoa collected by CT or EE showed no significant differences ( P > 0.05). In conclusion, the results obtained in the present study indicate that good quality freezable semen can be collected from cats by urethral catheterization after medetomidine administration. This new method of semen collection appears very useful in practice and, compared with the electroejaculation protocol, permits to obtain semen samples characterized by a higher concentration of spermatozoa, lower total volume and lower pH.