CNP, the third peptide of the mammalian natriuretic peptide family, exerts its biological effects through activation of the guanylyl cyclase B (GC-B) receptor and resulting cGMP accumulation. Our previous studies reported that CNP stimulates phosphorylation of MAPK family proteins in GH3 somatotrophs. This present study aimed to further characterise the mechanism of CNP-stimulated ERK phosphorylation and resulting biological consequences in GH3 cells. Western blot analyses showed CNP, but not ANP, stimulated ERK phosphorylation in a time- and concentration-dependent manner. Subsequent comparison of cGMP and phospho-ERK concentration response curves to CNP suggested the concentration of CNP required to stimulate ERK phosphorylation was two orders of magnitude lower (10−10 M) than that required to stimulate cGMP accumulation (10−8 M). Moreover, time-response studies using 10−3 M and 10−5 M dibutyryl cGMP failed to mimic the stimulatory effects of CNP on ERK phosphorylation, indicating a possible cGMP-independent mechanism. CNP stimulated phosphorylation of upstream kinases (C-Raf and MEK1/2) and downstream (p90RSK, CREB, Elk-1) targets of ERK pathway. Pharmacological inhibition of MEK1/2 (UO126) or Src (PP2) blocked the CNP effect on ERK phosphorylation, implying partial Src kinase-dependency. GC-B silencing by SiRNA abolished CNP-mediated phosphorylation of ERK and p90RSK, and blocked CNP-induced MKP-1 expression, whereas scrambled RNA failed to alter the CNP response. Interestingly, the SiRNA targeting resulted in loss of expression of GC-B1 and GC-B2 splice variants, implicating both of these receptors in mediating the CNP effects. Finally, in GH3 cells transiently transfected with the human αGSU promoter, CNP concentration-dependently stimulated promoter activity in a MEK-dependent manner. Collectively, these data suggest CNP-stimulated ERK phosphorylation requires either GC-B1 or 2 and Src kinase activity, but is cGMP independent. This clearly represents novel signalling events from these receptors. Funded by a BBSRC Project Grant (BBD0015601).