Using electrophoretic and immunoelectrophoretic methods we had previously characterized three major serum proteins of the lamprey, Petromyzon marinus: SDS-I, CB-111, and AS. SDS-I is present in highest concentration at the upstream migration period of the life cycle, whereas AS is present in highest concentration at the larval (ammocoete) period. Since the liver has been shown to be the major site of serum protein synthesis in many vertebrates, it may be assumed that the lamprey liver also engages in the same activity. In the present work we present evidence that the three major serum proteins of the lamprey are indeed synthesized by the liver. Liver slices from the larval and upstream migrant period were separately incubated with [35S]-methionine. After 2 or 3 hours of incubation, Triton X-100-soluble material was extracted from the liver slices and examined by crossed immunoelectrophoresis (CIE) and autoradiography. When extracts from the larval liver were run with antiserum made against larval serum, autoradiograms of the CIE patterns showed that AS and CB-I11 had been synthesized; the synthesis of AS was confirmed by the use of monospecific antiserum against AS. There was no indication of SDS-I synthesis during the incubation period. When extracts from the upstream migrant liver were run with antiserum made against whole serum from upstream migrants, autoradiograms of the resulting CIE patterns showed that SDS-I and CB-III had been synthesized; when antiserum against AS was used for the CIE, autoradiograms showed that AS had not been synthesized during the incubation period. Thus the differences in concentration of AS and SDS-I in the different periods of the life cycle may be due to differences in the synthetic capabilities of the liver at these periods.