The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1β, P2α and P2β, which form two heterodimers, P1α/P2β and P1β/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1β-P2β heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1β/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2β-P0-P1β/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.