Conventionally inseminated spermatozoa suffer a dramatic reduction in numbers during their long journey until fertilization. In addition sperm survival seems to be strongly affected by the reconstitution of the female reproductive tract in the post partum period. The purpose of this study was to develop a novel AI technique for cattle that allows the deposition of spermatozoa directly into the ampulla in the immediate vicinity of the fertilization site. This new reproductive biotechnique was investigated with focus on semen origin, sperm dosage, semen preparation and time of insemination. Finally, a first practical application was carried out by inseminating superovulated heifers with sex-sorted semen. In total, 49 Simmental heifers were used for 65 intratubal inseminations (ITI) with single ovulation and 8 ITIs after superovulation, respectively. Insemination into the oviduct was performed under epidural anesthesia via transvaginal endoscopy using a curved glass capillary loaded with semen. Two days later the oviduct and the adjacent uterine horn were endoscopically flushed and embryos or unfertilized oocytes were collected for determination of fertilization success. Across all experimental groups, tubal insemination successfully resulted in the collection of embryos; however, first tubal AI attempts and ITIs close to ovulation led to low recovery rates. In total, 109 complexes were flushed from ITIs in superstimulated heifers (n = 8) using sex sorted semen, of which 24 (22%) were at the embryo stage. In conclusion, it was shown that intratubal insemination can be successfully used for semen deposition, thus bypassing the lower female genital tract. Factors such as time of insemination, semen processing and semen quantity for superovulatory use should be further investigated.