We have investigated the effect of placing phosphoserine at the N-cap, N1, N2, N3, and interior position in alanine-based α-helical peptides. Helix contents of each peptide were measured by CD spectroscopy and titrations performed to determine pKa values. Data were analyzed with modified LifsonRoig theory to determine helix-coil parameters (n, n1, n2, n3, and w) and free energy changes for phosphoserine at each helical position. Results are given for a -1 and -2 phosphoserine charge state. Results show that phosphoserine stabilizes at the N-terminal positions by as much as 2.3 kcal·mol-1, while destabilizes in the helix interior by 1.2 kcal·mol-1, relative to serine. The rank order of free energies relative to serine at each position is N2 > N3 > N1 > N-cap > interior. Moreover, -2 phosphoserine is the most preferred residue known at each of these N-terminal positions. Experimental pKa values for the -1 to -2 phosphoserine transition are in the order N2 <N-cap <N1 <N3 <interior. This order agrees well with electrostatics calculations carried out with phosphoserine at the N-terminal positions and interior positions. Combining these with calculations at the C3, C2, C1, and C-cap positions gives results for phosphoserine along the length of the helix. We see a transition from phosphoserine stabilization at the N-terminus to destabilization at the C-terminus and can explain this in terms of the balance of protein solvation, favorable interactions, and dehydration. These results give insight into the phosphorylatable control of biological systems through positive or negative changes in stability.