In vivo methylation and demethylation processes were simultaneously investigated in freshwater tilapia after dietary exposure to mercury ((198)Hg(II) and methyl(200)Hg). During one month dietary exposure followed by two month depuration, both MeHg and THg increased continuously in muscle tissues but decreased in liver during depuration, indicating the inter-organ transportation of MeHg from liver toward muscle. Direct evidence of in vivo net methylation process in freshwater tilapia was observed. Specifically, 0.67-1.60% of the ingested Hg(198)(II) was converted into Me(198)Hg and deposited in fish muscle at the end of depuration. The methylation potential in terms of methylated fraction was elevated at higher temperature and decreased at higher dosage. However, no direct evidence of MeHg demethylation was observed. In contrast to some previous reports of dose-dependent demethylation, the percentage of MeHg in the liver decreased significantly with increasing THg concentrations, likely due to the faster inter-organ MeHg transportation from liver toward muscle. Our study demonstrates the important role of organ- and species-specific biodynamics in understanding mercury transformation and speciation in fish. The observed in vivo methylation process in tilapia was slow, suggesting that the high %MeHg in fish should be mainly derived from MeHg ingestion instead of in vivo transformation.