Radioimmunoassay (R1A) was utilized to determine serum immunoreactive testosterone (iT) concentrations in 5 adult male killer whales during sampling periods which varied for each whale and ranged from 1 to 10 years. The age of the animals sampled during the study ranged from 10 to 29 years. Blood was collected for serum from the animals during a voluntary fluke presentation behavior twice monthly to monthly during their individual sampling period. Samples (50 ul) were extracted twice with 5 ml diethyl ether prior to RIA analysis. Each sample was analyzed in duplicate and corrected for an extraction efficiency of 86 ± 4.4 %. The minimum concentration per tube which was distinguishable from zero was 3.4 pg Cross reactivities of the testosterone antiserum with other steroids were; 18.75 % for 5a-dihydrotestosterone; 3.0% for 5a-androstenediol; <1.0 % for androstenedione; and <0.1 % for all other steroids examined (progesterone, estradiol, estrone etc.). Serial diluted testosterone standards (1 to 1000 pg) exhibited parallel inhibition curves when compared to serial diluted pooled killer whale serum (approximately 470 pg/ml). Further characterization of the assay involved measurement of known amounts (5, 10, 25, 50, 100, 250, and 500 pg) of testosterone in 50 ul of charcoal-stripped plasma [Y=-6.14± 1.13X; Y= amount of testosterone added (pg.); r²=0.8937]. Inter assay and intra-assay coefficients of variation were 9.3 and 11.7 %, respectively. The mean and standard deviation (sd) concentration of iT in all ofthe killer whales was 1819.3 ± 1278.0 pg/ml. (n = 603; Range 6679 to 117 pg/ml). The mean and sd iT concentration during each month ranged from a low in October of 1418.46154 + 905.788 pg/ml (n=39; range 201 to 3464 pg/ml) to a high in April of 2235.42 + 1357.29 (n=50; range 321 to 5186 pg/ml). For all the animals, March through July were the only months that had significantly (Student -Newman-Keels multiple comparison (SNK), p<0.05) higher iT concentrations than the lowest iT concentration month of October. However, no significant differences (SNK, p<0.05) were observed in iT concentrations when all months accept October were compared to each other. No significant correlation was observed between iT concentrations and animal age (r2 = 0.02) or month (r2 = 0 004). All five whales sired calves during their sampling period. The age of the males when conception occurred ranged from 10 to 29yr. (n= 13; mean= 18.1 ± 5.6yr.). The mean iT concentration during the month that conception occurred was 2115.7 + 1581 sd (n = 12; range 275 to 4979 pg/ml). Conceptions occurred from Jan to Sept with 69.3% of the conceptions (46% in June) occurring during the period from March through July. The data validates the ability to determine iT concentrations in male killer whales and provides the first information concerning male killer whale reproductive endocrinology.