On two-dimensional electrophoresis gels, six protein spots from cotton ovules and fibers were identified as heat shock cognate 70 kD protein (Hsc70). Three spots corresponded to an experimental molecular weight (MW) of 70 kD (spots 1, 2 and 3), and the remaining three spots corresponded to an experimental MW slightly greater than 45 kD (spots 4, 5 and 6). Protein spots 1, 2 and 3 were abundant on gels of 0-day (the day of anthesis) wild-type (WT) ovules, 0-day fuzzless-lintless mutant ovules and 10-day WT ovules but absent from gels of 10-day WT fibers. Three individual transcripts encoding these six protein spots were obtained by using rapid amplification of cDNA ends (RACE). Edman degradation and western blotting confirmed that the three 45 kD Hsc70 protein spots had the same N-terminal, which started from the T271 amino acid in the intact Hsc70 protein. Furthermore, quadrupole time-of-flight mass spectrometry analysis identified a methylation modification on the arginine at position 475 for protein spots 4 and 5. Our data demonstrate that site-specific in vivo N-terminal truncation of the Hsc70 protein was particularly prevalent in cotton fibers, indicating that post-translational regulation might play an important role in cotton fiber development.