Gulf toadfish (Opsanus beta) are exceptionally capable of switching from excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses across the gill. Previous studies suggest that these urea pulses may be used for intraspecific chemical communication. To determine whether pulsatile urea excretion communicates reproductive status, toadfish were sexed using ultrasound and delivered conspecific-conditioned seawater (CC-SW) that previously housed a conspecific of the opposite sex, a conspecific chemical alarm cue (avoidance control), or a prey cue (attraction control). Swim behavior, attraction to or avoidance of the cues, and changes in the pattern of pulsatile urea excretion were monitored during and after delivery. Gulf toadfish did not spend more time in zones that were delivered CC-SW or prey cue. However, male toadfish spent significantly more time swimming after the delivery of female cues than control seawater (SW). In contrast, toadfish did not appear to have an immediate avoidance response to the conspecific alarm cue. Additionally, significantly more toadfish pulsed within 7 h of CC-SW and prey cue delivery compared to control SW, and pulse frequency was 1.6 times greater in response to CC-SW than control SW. These results, in combination with increased urea production and excretion the during breeding season, suggest that toadfish may use pulsatile urea excretion to communicate with conspecifics when exposed to chemosensory cues from the opposite sex.