Free endogenous methylarginines, Nω-monomethyl-L-arginine (L-NMMA) and Nω,Nω′-dimethyl-L-arginine (ADMA), inhibit NO synthases (NOSs) and are metabolized by dimethylargininedimethylaminohydrolase (DDAH). A postulated metabolism has been shown several times for DDAH-1, but the involvement of DDAH-2 in the degradation of ADMA and L-NMMA is still a matter of debate. Determination of the isoform-specific DDAH protein expression profiles in various porcine tissue types shows a correlation of DDAH activity only with DDAH-1 levels. DDAH activity (measured as L-citrulline formation from the conversion of methylarginines and alternative DDAH substrates) was detected in DDAH-1-rich porcine tissue types, that is, kidney, liver, and brain, but not in DDAH-2-rich porcine fractions, that is, spleen and thyroid. Furthermore, several ex vivo studies showed DDAH activity to be important for L-citrulline formation in porcine tissue and indicated the absence of an endogenous DDAH inhibitor in porcine tissue. This study provides new insights into tissue distributions as well as substrate selectivity for both DDAH isoforms. Although DDAH-1 is known to metabolize the endogenous NOS inhibitors L-NMMA and ADMA, a physiological function for DDAH-2 has yet to be determined. Hence, determining DDAH activity by methylarginine conversion is not suitable for analyzing isoform selectivity of DDAH-1 inhibitors as postulated.