The 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways. Here, using two-hybrid assays and in vitro assays of protein interaction, we show that the epsilon isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). Coprecipitation studies demonstrated an IGFI-dependent in vitro interaction between 14-3-3-glutathione S-transferase proteins and the IGFIR. In similar studies no interaction of 14-3-3 with the IR was observed. We present evidence to suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggested that the 14-3-3 interaction was dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region which has been implicated in IGFIR-dependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 was observed with a kinase-inactive IGFIR in the two-hybrid assay nor was any in vitro interaction with unstimulated IGFIR derived from mammalian cells. We show that the interaction of 14-3-3 with IRS-1 also appears to be phosphoserine-dependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation. In summary, our data suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR and within the central domain of IRS-1. The potential functional roles which 14-3-3 may play in IGFIR and IRS-1-mediated signaling remain to be elucidated.