The Transacetylase function of Calreticulin (CR) catalyzing the transfer of acetyl groups from acetoxycoumarins (AC) to certain proteins was identified for the first time in our laboratory. Protein acetyltransferase action of CR was termed Calreticulin Transacetylase (CRTAase). In the present work, CRTAase of rat tracheal smooth muscle cells (TSMC) was characterized with respect to the specificity for various AC and its role in the activation of nitric oxide synthase (NOS). 7,8-Diacetoxy-4-methylcoumarin (DAMC), a model AC, when incubated with TSMC along with L-arginine caused profound activation of NOS as compared to that with L-arginine alone. Further, the inclusion of N-omega-nitro-L-arginine methyl ester (L-NAME) along with DAMC resulted in the reduction of NO levels of TSMC to that of control, there by confirming the activation of TSMC NOS. Also, several AC were found to activate TSMC NOS in tune with their specificities to CRTAase. The results presented in this paper bear evidence for the activation of TSMC NOS by AC and their effectiveness to enhance NO of airway cells may be expected to find useful applications in respiratory diseases.