Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity. Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques. This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants. However, until now, only limited structural data for Bet v 1 have been published. Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F. D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W. R., Valent, P., Kungl, A. J., Breitenbach, M., Kraft, D., and Scheiner, O. (1993) J. Biol. Chem. 268, 19574-19580). Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry. Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5'-terminal primer and a nonspecific 3'-terminal primer or by immunological screening of a birch pollen cDNA library. These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938) is now referred to as Bet v 1a. High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen. Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.