Proteins that shuttle between nucleus and cytoplasm are implicated in transport and signal transduction processes. Using assays based on interspecies heterokaryons and microinjection of Xenopus oocytes, we examined what structural features determine nuclear export of shuttling proteins. Three classes of proteins were studied: first, wild-type and mutant forms of nucleolin, one of the first shuttling proteins identified; second, artificial nuclear reporter proteins derived from cytoplasmic pyruvate kinase; and third, wild-type and mutant lamins differing in their abilities to be incorporated into the lamina. Our results show that a protein does not require positively acting export signals to be transported from nucleus to cytoplasm; instead, its shuttling ability is limited primarily by intranuclear interactions. We conclude that nucleocytoplasmic shuttling is a general phenomenon not restricted to proteins involved in nucleocytoplasmic transport.