The cytoplasmic domain of the beta subunit of the insulin-like growth factor I receptor (amino acids 936-1337) was overexpressed in Sf9 insect cells using a baculovirus expression system, and the 6-His tagged receptor was purified by metal-affinity chromatography. Autophosphorylation of the receptor was concentration dependent, consistent with a trans phosphorylation mechanism. Phosphoamino acid analysis of the autophosphorylated receptor showed predominantly phosphotyrosine, but phosphoserine and phosphothreonine were also present. However, when the receptor was further purified by gel filtration on Sephadex G-100 and then autophosphorylated, phosphoamino acid analysis showed only phosphotyrosine. We conclude that the IGF-I receptor tyrosine kinase is not a dual-specificity kinase and that autophosphorylation of the beta subunit is by a trans mechanism.