A new nuclear substrate (N-60) for phosphatase 1 and 2Ac has been described. In contrast to nucleolin (C23), to which it is structurally and immunologically related, N-60 becomes dephosphorylated to 51% and 41% by phosphatases 1 and 2Ac, respectively, within 10 min. Incubation up to 20 min led to a complete dephosphorylation of N-60. The two other phosphatases tested (2B and 2C) did not dephosphorylate protein N-60 to the same extent as phosphatases 1 and 2Ac. In the case of nucleolin only 18% phosphate was released by all four phosphatases tested. The activity of both phosphatases, 1 and 2A, could be blocked by tumour promoter okadaic acid (100 nM) when N-60 was used as a substrate. These results support the notion that the observed okadaic-acid-induced hyperphosphorylation of N-60 in intact human fibroblasts may be caused by specific inhibition of phosphatases involved in the process of rDNA transcription.