Three fluorescent analogues of sphingomyelin (SPM), each containing pyrene in the fatty acyl residue, were synthesized and employed for the study of their mode of uptake by, and degradation within, intact cultured human skin fibroblasts. These were prepared by condensing sphingosylphosphocholine and the following fatty acids: pyrenedodecanoic acid (P12), pyrenesulphonylaminoundecanoic acid (PSA11) and pyrenepropenoic acid (P3:1). The cell association and catabolism of these SPM analogues by normal, Niemann-Pick-disease-Type-A and low-density-lipoprotein (LDL)-receptor-negative familial hypercholesterolaemia fibroblasts were investigated and compared with the metabolism of [cholinemethyl-14C]sphingomyelin. The catabolism of the fluorescent derivatives was monitored by measuring the appearance of the corresponding fluorescent ceramides. Two modes of uptake and degradation patterns were observed. Thus P12-SPM and radiolabelled SPM were taken up by LDL-receptor-mediated endocytosis when incubated with serum-containing medium, this conclusion being supported by the very low uptake by familial-hypercholesterolaemia fibroblasts, which lack the apolipoprotein-B/E receptor. After uptake, these compounds were metabolically degraded solely by the lysosomal sphingomyelinase, as evidenced by the fact that more than 98% of the SPM remained undegraded in Niemann-Pick-disease cells. By contrast, PSA11- and P3:1-SPMs were taken up by a receptor-independent endocytic pathway, as indicated by the similar rates of uptake in control and familial-hypercholesterolaemia cells in the absence or presence of fetal-calf serum in the culture medium. The degradation of PSA11-SPM and P3:1-SPM was brought about, in the main, by the lysosomal sphingomyelinase, but also by a yet uncharacterized process. The latter catabolic pathway, active in Niemann-Pick-disease-Type-A fibroblasts, seems to differ from the neutral Mg2+-dependent sphingomyelinase whose activity was undetectable in homogenates of skin fibroblasts. The present study emphasizes the influence of the structure of the fatty acyl moiety of SPM on its association with lipoproteins and/or cell membranes and on its intracellular routing and metabolic degradation.