Both thromboxane (TX) A2 and 8-epi prostaglandin (PG) F2α have been reported to stimulate mitogenesis of vascular smooth muscle (SM) in a number of species. However, TXA2 and 8-epiPGF2α mediated mitogenic signalling has not been studied in detail in human vascular SM. Thus, using the human uterine ULTR cell line as a model, we investigated TXA2 receptor (TP) mediated mitogenic signalling in cultured human vascular SMCs. Both the TP agonist U46619 and 8-epiPGF2α elicited time and concentration dependent activation of the extracellular signal regulated kinase (ERK)s and c-Jun N-terminal kinase (JNK)s in ULTR cells. Whereas the TP antagonist SQ29548 abolished U46619 mediated signalling, it only partially inhibited 8-epiPGF2α mediated ERK and JNK activation in ULTR cells. Both U46619 and 8-epiPGF2α induced ERK activations were inhibited by the protein kinase (PK) C, PKA and phosphoinositide 3-kinase inhibitors GF109203X, H-89 and wortmannin, respectively, but were unaffected by pertussis toxin. In addition, U46619 mediated ERK activation in ULTR cells involves transactivation of the epidermal growth factor (EGF) receptor. In humans, TXA2 signals through two distinct TP isoforms. In investigating the involvement of the TP isoforms in mitogenic signalling, both TPα and TPβ independently directed U46619 and 8-epiPGF2α mediated ERK and JNK activation in human embryonic kidney (HEK) 293 cells over-expressing the individual TP isoforms. However, in contrast to that which occurred in ULTR cells, SQ29548 abolished 8-epiPGF2α mediated ERK and JNK activation through both TPα and TPβ in HEK 293 cells providing further evidence that 8-epiPGF2α may signal through alternative receptors, in addition to the TPs, in human uterine ULTR cells.