Stathmin is a ubiquitous, highly conserved phosphoprotein which most likely acts as a relay integrating various intracellular pathways regulating cell proliferation, differentiation, and functions. At least 14 molecular forms of stathmin have been identified so far, which migrate as 2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) = 19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and whose pattern may reflect the state of activation of cells. We found that stathmin could be phosphorylated in vitro by at least three different protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase II, cAMP-dependent protein kinase catalyzed the phosphorylation of stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K). Interestingly, phosphorylation by both kinases together yielded all of the phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide analysis allowed us to demonstrate that the same four sites were exclusively found to be phosphorylated in vivo, in brain tissue as well as in control or nerve growth factor-stimulated PC12 cells. In this latter case, the major site phosphorylated in response to nerve growth factor being serine 25, it is likely that a kinase such as a mitogen-activated protein kinase, known to be activated by growth factors, might directly phosphorylate stathmin. The phosphopeptide map analysis allowed further identification of the specific combinations among the four sites whose phosphorylation is responsible for the characteristic two-dimensional polyacrylamide gel electrophoresis migration of the resulting stathmin forms both in vitro and in vivo and revealed the existence of likely structural interactions between the sites phosphorylated. In conclusion, our results show that phosphorylation of serines 16, 25, 38, and 63 accounts for all of the major functional stathmin forms observed in vivo. The present identification of these sites will foster a better understanding of some intracellular mechanisms involved in the diverse physiological regulation of the proliferation, differentiation, and functions of cells, including the role of stathmin in these processes as a relay integrating diverse signaling pathways.