To complete our investigation on the in vitro metabolism of 17b-Boldenone (b-BOLD), the biotransformation of b-BOLD and androsta-l,4-diene-3,17-dione (ADD) was studied . using primary bovine hepatocyte cultures incubated for 3,6 and 24 hours with 100uM of b-BOLD or 100uM of ADD or a combination of both (90 uM b-BOLD + 10 uM ADD). The metabolites were separated and identified by liquid chromatography-high resolution mass spectrometry. After 6 h of incubation with b-BOLD a significant amount was oxidized into ADD or transformed into hydroxylated metabolites. After 24 h, the production of hydroxylated (OH) metabolites of b-BOLD (6b-OH, 17b-BOLD, 6b-OH, 17a-BOLD and l 6a-OH,17a-BOLD) and ADD (6-0H-ADD) was confirmed; 17a-Boldenone (a-BOLD) was among the main metabolites detected, and ADD was found to a minor extent only. When culture cells were incubated with both b-BOLD and ADD, the metabolic pattern of BOLD prevailed. When ADD alone was added to celi cultures, after 6 hours the formation of b-BOLD was observed, a-BOLD was the main metabolite found after 24 h, and 6-0H-ADDs were also detected. As observed with other anabolic steroids, dihydroxylated metabolites of boldenone were also detected.