Very little is known about the attainment of puberty in reptiles. In the American alligator (Alligator mississippiensis) males are assumed to be sexually mature at about 1.8 m in total length, but it is not clear at what size they produce testosterone, spermatozoa and mate successfully. We re-examined this question by studying plasma testosterone levels in blood samples from a large sample of alligators (∼1,500) collected every month of the year and ranging in size from approximately 61 cm (2 ft) to 360 cm (11.5 ft). Testosterone values ranged from 0.05–115.41 ng/mL. All size classes of alligators exhibited a seasonal cycle in testosterone levels, but the concentrations were size-dependent: the larger the alligator the higher the testosterone. In all size-classes testosterone reached a peak in the breeding season (March–May). Mean testosterone in the largest size-class during breeding was 75 ng/mL whereas in the smallest size-class peak testosterone was less than 3 ng/mL. The smallest size-class (59–89 cm) showed an additional rise in testosterone in late summer. The attainment of sexual maturity in alligators appears to be closely associated with growth and is a gradual process lasting several years. Sexually immature alligators show a seasonal pattern of testosterone secretion similar to that of adults, but the values are significantly lower.