Extensive digestion of human serum albumin with trypsin at pH 8.8 yields essentially one main fragment which is resistant to further tryptic degradation. The fragment has been characterized by amino acid analysis, N- and C-terminal analyses, cyanogen bromide digestion, electrophoresis, ultracentrifugation and gelfiltration, and circular dichroism measurements. The results indicate that the main fragment consists of the amino acids 182-585. Repeated digestion did not degrade the isolated fragment further. The fragment mainly retains the secondary and tertiary structure of intact human serum albumin as well as its capacity to bind bilirubin and diazepam. The localization of the binding sites for these substances is discussed.