BACKGROUND Long term use of NSAIDS is mainly accompanied by major health implications such as gastrointestinal erosions, ulcerations and nephrotoxicity. These side effects arise from local irritation by the carboxylic acid moiety, that is common to most of NSAIDs (topical effect), in addition to decreased cytoprotective prostaglandin production. Therefore, in the medicinal chemistry research area, there is an ongoing need for the discovery of new, potent and safer anti-inflammatory lead compounds devoid of the irritant carboxylic acid moiety. METHODS A series of new 3-substituted-2-thioxo-thieno[2,3-d]pyrimidine derivatives were synthesized through reacting the starting 3-amino-2-thioxo-thieno[2,3-d]pyrimidines with different aromatic aldehydes. The structure of all newly synthesized compounds was confirmed with spectral and elemental analyses. The synthesized thieno[2,3-d]pyrimidines were investigated for in vivo anti-inflammatory activity, using the carrageenan induced paw edema test. The possible antiinflammatory mechanism was also evaluated by determining the concentration of prostaglandin E2 (PGE2) in blood serum using a rat specific PGE2 ELISA kit. RESULTS All test compounds could significantly reduce carrageenan induced paw edema comparable to diclofenac sodium as a potent anti-inflammatory drug. Moreover, they could decrease the concentration of PGE2 in blood serum. Interestingly, compound 4c exhibited the most potent in vivo anti-inflammatory activity with protection of 35%, 36% and 42% against carrageenan-induced paw edema after 1h, 2h and 3h, representing 92%, 86% and 88% respectively of diclofenac activity. It also decreased the concentration of PGE2 in blood serum to 19 pg/ mL which is comparable to diclofenac with PGE2 concentration of 12 pg/ mL. Moreover, Compounds 4f, 4a, 4i and 4e exerted significant anti-inflammatory activity after 4h, representing 71%, 69%, 63% and 61% respectively of diclofenac activity. Furthermore, they significantly decreased the concentration of PGE2 in blood serum. CONCLUSIONS These thienopyrimidines may be used as good candidates for the search of promising, potent and safe antiinflammatory leads for being free from acidic functions.