Evidence suggests overexpression of COX-2 and its role in many human cancers, including lung. However, the regulatory mechanism underlying COX-2 overexpression in lung cancer is not fully understood. We herein investigated whether COX-2 is overexpressed in human airway cancer cell lines, including A549 (lung), Hep-2 (bronchial), and NCI-H292 (alveolar). When grown in cell culture medium containing 10% FBS (serum), of note, there was strong and transient induction of COX-2 protein and mRNA in NCI-H292 cells, but little or low COX-2 expression is seen in A549 or Hep-2 cells. Interestingly, strong and sustained activities of ERK-1/2, JNK-1/2, p38 MAPK, and PKB were also shown in NCI-H292 cells grown in presence of serum. Profoundly, results of pharmacological inhibition studies demonstrated that the serum-dependent COX-2 up-regulation in NCI-H292 cells is attributed to not only the p38 MAPK-, PI3K/PKB-, and ERK-1/2-mediated COX-2 transcriptional up-regulation but also the p38 MAPK- and ERK-1/2-mediated post-transcriptional COX-2 mRNA stabilization. Of further note, it was shown that the ERK-1/2 and PI3K/PKB (but not COX-2, p38 MAPK, and JNK-1/2) activities are necessary for growth of NCI-H292 cells. These findings collectively demonstrate for the first time that COX-2 expression is transiently up-regulated by serum addition in NCI-H292 cells and the serum-induced COX-2 expression is closely linked to the p38 MAPK-, ERK-1/2-, and PI3K/PKB-mediated COX-2 transcriptional and post-transcriptional up-regulation.