Determinations of both the free and bound form of N-acetyl-neuraminic acid (NANA) in several human body fluids, such as serum, cerebrospinal fluid (CSF), saliva, urine, amniotic fluid, and milk were carried out by HPLC with fluorimetric detection. The method utilized 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) as a fluorimetric derivatizing reagent. Free-form NANA was obtained from the body fluids after ultrafiltration with Microcon 10 (YM-10 cellulose membrane, filtration limit M(r) = 10,000, Amicon). The DMB derivative of NANA was separated isocratically by a Nucleosil 5C18 column with a mixture of 0.1 M sodium phosphate buffer (pH 2.0)-methanol (75:25, v/v). A gradient elution system was used for urine analysis. Analysis times were 10-30 min. Recoveries of free NANA by ultrafiltration were satisfactory: 95.66 +/- 1.80% for serum and 97.27 +/- 1.55% for CSF, respectively. The high sensitivity and specificity render this method applicable to all the body fluids tested. Although a physiological role for free NANA has not yet been elucidated, the method presented promises to contribute to the basic understanding of the NANA metabolism.