Coenzyme A (CoASH) is an obligate cofactor for lipids undergoing beta-oxidation in peroxisomes. Although the peroxisomal membrane appears to be impermeable to CoASH, peroxisomes contain their own pool of CoASH. It is believed that CoASH enters peroxisomes as acyl-CoAs, but it is not known how this pool is regulated. The mouse nudix hydrolase 7 (NUDT7alpha) was previously identified in peroxisomes as a CoA-diphosphatase, and therefore suggested to be involved in regulation of peroxisomal CoASH levels. Here we show that mouse NUDT7alpha mainly acts as an acyl-CoA diphosphatase, with highest activity towards medium-chain acyl-CoAs, and much lower activity with CoASH. Nudt7alpha mRNA is highly expressed in liver, brown adipose tissue and heart, similar to enzymes involved in peroxisomal lipid degradation. Nudt7alpha mRNA is down-regulated by Wy-14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, in a PPARalpha-dependent manner in mouse liver. In highly purified peroxisomes, nudix hydrolase activity is highest with C(6)-CoA and is decreased by fibrate treatment. Under certain conditions, such as treatment with peroxisome proliferators or fasting, an increase in peroxisomal CoASH levels has been reported, which is in line with a decreased expression/activity of NUDT7alpha. Taken together these data suggest that NUDT7alpha function is tightly linked to peroxisomal CoASH/acyl-CoA homeostasis.