The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C(12:0)), N-myristoyl- (C(14:0)) and N-palmitoyl- (C(16:0)) sphingosine (C(18:1)) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C(18:0)) substrate. The activity towards N-linolenoylsphingosine (C(18:3)/C(18:1)), N-linoleoylsphingosine (C(18:2)/C(18:1)) and N-oleoylsphingosine (C(18:1)/C(18:1)) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C(18:0)/C(18:1)). The activity towards N-stearoyldihydrosphingosine (C(18:0)/C(18:0)) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C(6:0)/C(18:1)), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2-12)-fold and 7 (4-17)-fold higher K(m) values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.