The in vitro splicing of kappa immunoglobulin precursor mRNA was studied as an example of a naturally occurring mRNA possessing multiple 5' splice sites. Several kappa mRNAs were generated in vitro by using an SP6 transcription system and were spliced in nuclear extracts derived from HeLa cells. Products and intermediates resulting from in vitro splicing were identified and characterized. In contrast to the in vivo situation, in which apparently only the 5'-most splice donor site is used, all of the 5' splice sites were used in vitro with equal frequency. Neither the presence or absence of variable region coding sequences nor the deletion of intron sequences had an effect on in vitro splice site selection.