A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of josamycin residues in four porcine tissues (i.e., muscle, liver, kidney and fat). The sample preparation consisted of a homogenization step in an acetonitrile–10 mmol l^–1 phosphate buffer mixture, pH 6.0 (35 + 65), centrifugation and a liquid–liquid extractive clean-up of the resulting supernatant with isooctane. Pre-column derivatization of josamycin was performed using cyclohexa-1,3-dione in ammonium acetate buffer, pH 5.0 (90 °C for 2 h). The derivative was chromatographed in an isocratic reversed-phase HPLC system. A LiChrospher RP 18 end-capped (5 µm) column was eluted with an acetonitrile–methanol–10 mmol l^–1 phosphate buffer mixture, pH 6.0 (45 + 5 + 50). The capacity factor of the josamycin derivative was 17.5. Detection was achieved using spectrofluorimetry (λ_ex= 375 nm; λ_em= 450 nm). The structure of the derivative was assessed by using mass spectrometry. Full selectivity was obtained in the HPLC system versus other macrolide antibiotics (tylosin, spiramycin and erythromycin), aldehydes (formaldehyde, acetaldehyde and benzaldehyde) and endogenous compounds. Linearity and repeatability were tested. Correlation coefficients, for callibration curves in the range of 0.1–3.2 µg g^–1, were greater than 0.999 for all tissues and the relative standard deviation (s_r) was 4.9%(1.6 µg g^–1; n= 6); recovery was higher than 88%.