The objectives in this study were to characterize the pattern of testosterone secretion over 24 h in the captive Australian sea lion and to establish whether exogenous hormone stimulation could be used to obtain an index of testosterone secretion in this species. Blood samples were collected from three males at 3-h intervals for 24 h on three occasions at 2-week intervals. Exogenous GnRH, hCG and LH were administered at the end of each 24-h sampling period and changes in circulating testosterone monitored over 5 h. Circulating concentrations of testosterone during 24-h sampling periods were greater (P < 0.01) during darkness than during daylight. Exogenous hCG administered induced a significant rise in circulating concentrations of testosterone. In the 5 h following GnRH, hCG and LH administration, circulating testosterone concentrations reached similar levels to the highest concentrations observed during the respective 24-h sampling periods. These findings have provided evidence of a 24-h pattern of testosterone secretion in the Australian sea lion. This study has also shown that the testis response to exogenous GnRH, hCG and LH in the Australian sea lion can provide an index of the prevailing testosterone biosynthetic capacity of the testes.