Fusion proteins for cell surface expression in the yeast Saccharomyces cerevisiae were constructed that consisted of the N-terminal leader sequence of Kre1p, followed by the nine amino acid viral epitope hemagglutinin (HA), and the carboxyterminal anchoring domain of either Cwp2p or Flo1p. All fusions were constitutively expressed under transcriptional control of the phosphoglycerate kinase promoter and immunofluorescence analysis indicated that in each construct the HA peptide was correctly anchored to the outer yeast cell surface. Successful solubilization of the cell wall fusions by laminarinase treatment indicated that the fusions are covalently linked to cell wall beta-1,3-D-glucans in vivo. FACS analyses further demonstrated that 70% of the yeast cell population expressed the corresponding cell wall fusion. Neither the number of positive cells within the population nor the distribution of the fusion at the single-cell level were negatively affected by replacing the "heterologous" Kre1p leader by the "native" Cwp2p leader. Insertion of a 350 amino acid Ser/Thr-rich spacer sequence into the fusions led to a dramatic increase in HA peptide accessibility on the yeast cell surface. Our data show that FACS analyses represent a valuable means for investigating cell surface expression, and indicate that artificial-spacer-elongated cell wall fusions might raise novel possibilities for cell surface expression of heterologous proteins in yeast.