The endosomal sorting complex required for transport (ESCRT) protein machinery comprises four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, that facilitate receptor sorting into the lumen of multivesicular endosomes (MVEs) in order to terminate signalling receptors for final degradation within the lysosomes. Even though ESCRT proteins appear to be essential for the biogenesis of MVEs in Saccharomyces cerevisae, it is not clear whether ESCRT-independent pathways for MVE biogenesis exist in higher organisms. In this study we maximized inhibition of ESCRT-dependent pathway by depleting cells of key subunits of all four ESCRTs and followed MVE formation and epidermal growth factor (EGF) receptor (EGFR) traffic using electron and confocal microscopy. There was a dramatic alteration in the morphology of components of the endocytic pathway in ESCRT-depleted cells, but early and late endosomes stayed clearly differentiated. Importantly, although EGF-induced formation of MVEs was highly sensitive to ESCRT depletion, EGF-independent formation of MVEs could still occur. The MVEs remaining in ESCRT-depleted cells contained enlarged intralumenal vesicles into which EGFRs were not sorted. Our observations suggest that both ESCRT-dependent and ESCRT-independent mechanisms of MVE biogenesis exist in mammalian cells.