The number of superior cervical ganglion (SCG) neurons does not differ for males and females on the day of birth, but by 15 days, after most of the normal neuron death has occurred, males have significantly more neurons than females. This difference persists in the adult. The present study was undertaken to determine whether the presence of afferent input to the SCG is required for the development of this sex difference. Bilateral transection of the cervical sympathetic trunk, which deafferents the SCG neurons, or a sham operation was performed on male and female Sprague Dawley rats on the day of birth. Numbers of neurons were counted in SCGs of animals sacrificed on either postnatal day 4 or 15, before or after normal development of the SCG sex difference. At 4 days, the number of SCG neurons in sham-operated males and females were not different, but by 15 days, females had lost a significant number of neurons, whereas the males had not. Transection of the cervical sympathetic trunk led to a significant loss of over 6,000 SCG neurons by postnatal day 4 in both males and females. Whereas some of this loss is due to axotomy of caudally projecting SCG neurons, at least half of the neuron loss is due to removal of the afferent input. At 15 days, sham-operated males had significantly more SCG neurons than did sham-operated females, but the gender difference was not significant in animals with neonatally deafferented ganglia. Thus, the normal development of the gender difference in SCG neuron numbers requires an intact afferent input.