Since the late 1980s, elevated levels of mercury have been reported in the tissues of the Florida panther (Puma concolorcoryi) from the Florida Everglades. The extent, degree, and length of time of mercury contamination in the Florida panther are unknown. The objective of this study was to determine the historical and other patterns of monomethyl and inorganic mercury in the Florida panther by analysis of mercury in panther hair from museum collections. In addition, this study evaluated the effects of preservation of skins on mercury concentrations in hair and the representativeness of museum collections for evaluating historical trends of contamination in the Florida panther. Hair from 42 Florida panther specimens collected from 1896 to 1995 was analyzed for both monomethyl and inorganic mercury. Monomethyl mercury (MMHg) and inorganic mercury (IHg) were found in all specimens. Monomethyl mercury in hair from untanned skins was significantly higher than MMHg in hair from tanned skins. For untanned specimens, the mean MMHg concentration in hair was 1.62 ± 1.87 μg/g (range 0.11 to 6.68 μg/g, n = 16). Monomethyl mercury accounted for 88% of the total mercury in untanned Florida panther hair. No sexual or geographical differences were found. Although MMHg is generally stable in hair, the tanning process appears to reduce the amount of MMHg in hair. In addition, exogenous IHg contamination of the panther hair was found in museum specimens, especially in older specimens. The implication of these and other factors in interpreting results of museum studies is discussed. The presence of MMHg in panther hair since the 1890s indicates long-term and widespread exposure of the Florida panther to mercury. Levels of MMHg are significantly greater in the 1990s than the 1890s. When combined with field studies of mercury in the Florida panther, considerable individual variability is observed, reflecting short-term changes in exposure of individual panthers to mercury. Although museum specimens showed a significant increase in MMHg over the last 100 years, they did not show the magnitude of increase that field populations of Florida panthers did. A number of Florida panthers appeared to be at risk from mercury over their lifetimes, especially individuals from the early 1990s.