We have investigated the cell-specific activity of the GT-IIC, GT-I, Sph-II, Sph-I, and octamer motifs of the SV40 enhancer in four cell lines (HeLa cells, MPC11 plasmocytoma B cells, and non-differentiated and retinoic acid-differentiated F9 embryonal carcinoma cells). Our present results reveal the existence of three classes of motifs that interact with cell-specific enhancer factors but that have no enhancer activity of their own: those (class A) that generate enhancer activity following oligomerization of tandem repeats of the motif; those (class B) that cannot enhance transcription when oligomerized as a tandem repeat but whose association with a second motif results in transcription activation after oligomerization; and those (class C) that exhibit enhancer activity when a single copy of the motif is oligomerized. Three levels of functional organization of enhancers can also be defined. The possible implications for enhancer functions are discussed.