A technique was developed to assess the number of cock spermatozoa penetrating the perivitelline layer (PL) in oviposited eggs in vivo. Two trials were conducted to test this technique and to establish correlation values between fertility and sperm penetration (SP). First, three Athens Canadian Randombred males, previously tested as having high fertility (100%), were each housed with seven hens. Sperm penetration was determined from eggs laid over a 3-d period (n = 41) with the mean number of spermatozoa penetrating the PL overlying the germinal disc (GD; 1.35 mm2 area) and nongerminal disc (NGD) areas being 162.8 and 8.4, respectively. Following removal of the males, SP was monitored to establish its duration with an average of 4.6 eggs analyzed per male per day. Mean sperm penetration during this period declined from 167.0 to .2 and from 9.2 to 0 for the GD and NGD regions, respectively. The mean duration of SP was 15.7 and 11.3 d for the GD and NGD PL, respectively. The duration of fertility was also established to be 14.0 d. There was a positive correlation between sperm penetration of the GD PL and fertility from eggs laid by naturally mated hens (r = .89, P < .001). In the second trial, three groups (1, 2, or 3) of 16 hens (35 wk of age) each were artificially inseminated weekly for 4 consecutive wk with either 100, 50, or 25 million sperm/50 microL, respectively. Inseminations were repeated weekly for 12 consecutive wk. Mean values were obtained from each of three 4-wk periods and used as replicates. Mean SP values from the GD PL for Groups 1, 2, and 3 were 402, 19.5, and 14.1, with fertility values of 95.8, 92.4, and 83.3%, respectively. Each replicate mean was obtained from approximately 24 eggs per group per day postinsemination. A significant correlation between SP of the GD PL and fertility (r = .90, P < .001) was established using artificial insemination of hens.