The purpose of this investigation was to determine whether there has been a change in the human blood concentration of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and five other fluorochemicals since 1974. Blood samples were collected in 1974 (serum) and 1989 (plasma) from volunteer participants of a large community health study. The study included a total of 356 samples (178 from each time period). These samples were analyzed by high-pressure liquid chromatography/tandem mass spectrometry methods. The median 1974 and 1989 fluorochemical concentrations, respectively, were as follows: PFOS, 29.5 ng/mL vs. 34.7 ng/mL; PFOA, 2.3 ng/mL vs. 5.6 ng/mL; perfluorohexanesulfonate (PFHS), 1.6 ng/mL vs. 2.4 ng/mL; and N-ethyl perfluorooctanesulfonamidoacetate (PFOSAA), less than the lower limit of quantitation (LLOQ; 1.6 ng/mL, vs. 3.4 ng/mL). For N-methyl perfluorooctanesulfonamidoacetate (M570), perfluorooctanesulfonamide, and perfluorooctanesulfonamidoacetate, median serum concentrations in both years were less than the LLOQ values (1.0, 1.0, and 2.5 ng/mL, respectively). Statistical analysis of 58 paired samples indicated that serum concentrations of PFOS, PFOSAA, PFOA, PFHS, and M570 were significantly (p < 0.001) higher in 1989 than in 1974. The data from 1989 were then compared with geometric mean fluorochemical concentrations of serum samples collected in 2001 from 108 American Red Cross adult blood donors from the same region. Except for M570, there were no statistically significant (p < 0.05) geometric mean fluorochemical concentration differences between the 1989 and 2001 samples. In conclusion, based on this study population, PFOS and other serum fluorochemical concentrations have increased between 1974 and 1989. Comparison with other regional data collected in 2001 did not suggest a continued increase in concentrations since 1989.