To determinate the area of the epididymis that yields spermatozoa most capable of supporting embryonic development. During the last 9 years, 46 men affected by obstructive azoospermia (OA) consented to have spermatozoa surgically retrieved from three areas of the epididymis: caput, corpus, and cauda. Sperm characteristic were analyzed and compared among the retrieval sites that were used for ICSI with their female partners. Clinical outcomes were compared among the different sources of spermatozoa. Consenting men underwent epididymal sperm retrieval, with specimens evaluated according to WHO 2010 criteria. A total of 36 specimens retrieved from the caput, 7 from the corpus, and 3 from the cauda were used for ICSI, controlling for maternal age (≤37 years old). Fertilization, implantation, and delivery rates were assessed and compared for all three areas of the epididymis. Unpaired t and Fisher’s exact tests were used to compare outcomes. A P value of <0.05 was considered significant. A total of 46 men were divided into three groups according to the spermatozoa source used: caput, corpus, and cauda. Paternal age was comparable among the three groups (39.7± 12). Average and standard deviation of spermatozoa concentration was 35.1± 39 in the caput, 40±29 in the corpus, and 30.2± 59 x106/ml in the cauda. The mean motility was 15.8±15% in the caput, rising to 40.1±29% in the corpus (P<0.01), and decreasing to 7±12% in the cauda (P<0.05). The mean numbers of injected oocytes were comparable at 12.7, 11, and 12.5, for the caput, corpus, and cauda, respectively. The fertilization rate with specimens from the caput was 65.8% (303/460), it rose to 77.6% (59/76) with specimens from the corpus (P<0.05), and reached the highest level at 88% (44/50, P<0.05) with spermatozoa from the cauda. The proportion of high-quality embryos was 73.5% (25/34) from sperm retrieved from the caput, 75% (3/4) from the corpus, and reached 100% (3/3) from the cauda. There was a trend of increasing implantation rates through the regions of the epididymis. With specimens retrieved from the caput, the implantation rate was 34.8% (23/66), it rose to 44.4% (4/9) from the corpus, and increased to 57.1% (4/7) from the cauda. The same trend was observed for delivery rates. In cycles using caput spermatozoa, the delivery rate was 48.4% (16/33) with 5.8% (1/17) pregnancy loss. In cycles using spermatozoa from the corpus, the delivery rate was 75% (3/4) with no pregnancy loss. Ultimately, the cycles with cauda had the highest delivery rate, with 100% (3/3). This study supports the current knowledge that while progressing through the epididymal regions, testicular spermatozoa undergo structural and metabolic changes that translate in phenotypic and functional processes. During the epididymal journey, spermatozoa appeared to enhance their ability to support embryonic development and implantation. Although they have poorer kinetic characteristics, we have found that spermatozoa retrieved from the cauda epididymis yield higher fertilization and pregnancy rates. Larger studies are needed to confirm these findings.