Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.