Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100 : 28 : 27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met 5-enkephalin (N-terminal hydrolysis) at rates of 188 ± 38 and 420 ± 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39–437 pmol/min/mL) was closely correlated with that of Met 5 -enkephalin degradation (37–833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by the aminopeptidase inhibitors amastatin ( ic 50 = 20 nM), bestatin (4–7 μM) and puromycin (30–35 μM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The K m of Met 5 enkephalin for the CSF aminopeptidase activity was 201 ± 19 μM (N = 4). Rates of hydrolysis of the Tyr 1-Gly 2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu 5-enkephalin, dynorphin A fragments [1–7], [1–10] and [1–13] and dynorphin A at rates of 2.05 ± 0.27, 1.76 ±0.18, 0.94 ± 0.06, 0.55 ± 0.14 and 0.16 ± 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.