Catecholamines (CATs), the major regulator of lipolysis in adipose tissue, are produced mainly by the sympathoadrenal system. However, recent studies report endogenous CAT production in adipocytes themselves. This study investigated the effects of single and repeated (7–14 times) immobilization (IMO) stress on CAT production in various fat depots of the rat. Single IMO quickly induced a rise of norepinephrine (NE) and epinephrine (EPI) concentration in mesenteric and brown adipose depots. Adaptive response to repeated IMO included robust increases of NE and EPI levels in mesenteric and subcutaneous adipose tissue. These changes likely reflect the activation of sympathetic nervous system in fat depots by IMO. However, this process was also paralleled by an increase in tyrosine hydroxylase gene expression in mesenteric fat, suggesting regulation of endogenous CAT production in adipose tissue cells. Detailed time-course analysis (time course 10, 30, and 120 min) clearly showed that repeated stress led to increased CAT biosynthesis in isolated mesenteric adipocytes resulting in gradual accumulation of intracellular EPI during IMO exposure. Comparable changes were also found in stromal/vascular fractions, with more pronounced effects of single than repeated IMO. The potential physiological importance of these findings is accentuated by parallel increase in expression of vesicular monoamine transporter 1, indicating a need for CAT storage in adipocyte vesicles. Taken together, we show that CAT production occurs in adipose tissue and may be activated by stress directly in adipocytes.