A major goal of this study was to determine whether serum protein fractions of healthy Florida manatees differ with age, sex, or living environments (wild versus housed). A second goal was to determine which serum protein fractions vary in diseased versus healthy manatees. Serum protein fractions were determined using agarose gel electrophoresis. Healthy adults had slightly higher total serum protein and total globulin concentrations than younger animals. This largely resulted from an increase in gamma globulins with age. Total serum protein, albumin, alpha-1 globulin, beta globulin, and total globulin concentrations were slightly higher in housed manatees compared to wild manatees, but there was no significant difference in the albumin/globulin (A/G) ratio, suggesting a difference in hydration between these groups. No significant differences were attributable to sex or pregnancy. Serum albumin concentrations and A/G ratios were significantly lower for manatees with boat trauma, entanglements, emaciation, or cold stress compared to healthy manatees. Variable increases were seen in alpha-1globulins, alpha-2 globulins, beta globulins, and gamma globulins. These globulin fractions contain positive acute-phase proteins and immunoglobulins, and their increases may reflect acute or chronic active inflammation. Changes in serum protein fractions were not consistent enough to justify the use of serum protein electrophoresis as a routine diagnostic test for manatees. However, serum (or plasma) protein electrophoresis is required when accurate values for albumin and globulins are needed in manatees and in determining which protein fractions may account for a hyperproteinemia or hypoproteinemia reported in a clinical chemistry panel.