Break-down of branched-chain amino acids (BCAA) plays a major role in the metabolic adaptation to the increased energy needs due to lactation. We aimed to specify the potential role of various metabolically active tissues in BCAA catabolism of early-lactating dairy cows. Liver, muscle (M. semitendinosus), mammary gland (MG), subcutaneous (scAT; tail head) and visceral adipose tissue (vAT; omental fat) of 25 primiparous Holstein cows were collected on d 1, 42 and 105 postpartum (p.p.) during slaughter and examined for mRNA and protein abundance and the activity of the rate-limiting enzyme BCKDH. Target genes were quantified by qPCR, protein abundance was measured via Simple Western, and enzyme activity was determined spectrophotometrically. Data were analyzed using a Linear Mixed Model (fixed: tissue, time, tissue × time; random: cow; post-hoc: Bonferroni; SPSS). The mRNA abundance of the α subunit of the BCKDH E1 component (BCKDHA) in muscle was lower (P < 0.05) compared with those in liver, MG, and scAT. The mRNA abundance of the β subunit of the BCKDH E1 component (BCKDHB) was about 5-fold higher in liver than in any other tissue (P < 0.001). Both BCKDHA and BCKDHB mRNA in MG and vAT were affected by time (P < 0.05): Abundance on d 105 was on average twice as high as on d 1 p.p. (all P < 0.05 except for BCKDHB in vAT, P = 0.06). The BCKDHA protein abundance was greatest in liver and MG, followed by the 2 AT and then muscle (P < 0.001). At all 3 time points, the BCKDH activity was around 7-fold greater in liver compared with MG (P < 0.001). In liver, an increase of BCKDH activity from d 1 to d 42 p.p. was observed (P = 0.02), whereas no time-dependent changes were detected in MG. Muscle and AT did not show any measurable BCKDH activity. During early lactation, liver seems to be a physiologically significant site of BCAA catabolism, while the further involvement of muscle, beyond being the main supply of these AA especially in times of insufficient intake, remains unclear. The observed lower activity in MG indicates that BCAA might be spared from oxidation possibly for de novo protein synthesis.