Mitogen activated protein kinases (MAPK) are a family of ancient, highly conserved signalling molecules that control intracellular signal transduction. An acute response to extracellular stimuli transduced by the MAPK cascade leads to changes in gene expression and results in major developmental changes in organisms. While the role of MAPKs in cell adhesion, apoptosis, proliferation and survival is well studied, their role in sex determination has only recently been discovered. Loss of MAP3K4 has revealed its requirement in sex determination in the mouse, while mutations in MAP3K1 result in increased phosphorylation of downstream targets ERK1/2, increased binding of GTPase RhoA and is associated with sex reversal in humans. Using our unique and well-studied marsupial model, the tammar wallaby, we sought to identify the role of the MAPK pathway in gonadal sex determination. We hypothesise that MAPKs regulate granulosa and Sertoli cell fate and upsetting the MAPK cascade may cause an imbalance between testis and ovary promoting genes leading to sex reversal. We tested this hypothesis, using a gonadal explant culture system to analyse the effect of inhibitors of ERK1/2 (U0126) and RhoA (Y27632) on undifferentiated tammar gonads to determine if abrogation of MAPK affects gonadal morphology, Sertoli and granulosa cell fate and leads to a sex-reversed phenotype. Undifferentiated tammar gonads were cultured in the presence or absence of inhibitors in 1.5% agar moulds in media containing DMEM/10% FBS/ penicillin-streptomycin at 37°C, 5% CO₂. Immunohistochemistry was used to determine the localisation of testis promoting SOX9, FGF9 and ovarian marker β-cateninin order to identify any signs of sex reversal. MAP3K1, MAP3K4, ERK1/2 and RhoA are all expressed in both sexes before gonadal sex determination from the undifferentiated tammar gonad to at least 8 days post-partum. Our findings greatly contribute a deeper understanding of the MAPK pathway and its involvement in sex determination.