Integral membrane proteins have parts of their polypeptide backbones embedded in a phospholipid bilayer. These hydrophobic, membrane-spanning domains are separated by hydrophilic segments exposed to the aqueous environment and seem to fall into two basic modalities: a-helix bundle and anti-parallel/3-barrel membrane domains. For the helix bundle proteins, two easily identifiable features appear to be the major structural determinants: the long apolar stretches that form the transmembrane a-helices, and the biased distribution of charged residues in the polar regions, known as the'positive-inside'rule (von Heijne, 1986). Recently, attempts to predict topologies of eukaryotic and prokaryotic integral membrane proteins that form a-helical structures, have been reported (von Heijne, 1992; Sipos and von Heijne, 1993). This work has indeed shown that the topology of such proteins can be effectively deduced from the amino acid sequence. Thus, the aim of developing TopPred II is to compile all existing knowledge about topology in order to permit easy access to prediction of membrane protein topologies. TopPred II is an improved version of the preceding freeware TOP-PRED (von Heijne, 1992). It is now a stand-alone application written in Symantec THINK Pascal to allow its operation on any Macintosh computer with a 6.0. 2 (or beyond) system, respecting the standard Apple file-and window-handling procedures, and making extensive use of the graphic abilities of Macintosh computers. The compiled program is very compact (~ 90 kbytes) and all the default parameters, scales and texts have been built into resources to allow easy access and ability for permanent modifications by the user. Input sequence files are limited to 2000 amino acids. Sequences can be handled one by one or in groups of up to 20. Parameters and scales can be easily modified through standard dialogs, which also enable one to re-establish the TopPred II default values. Calculation of the hydrophobicity profile, transmembrane segments and topologies (see below) can be requested. All the displayed results (texts as well as graphics) can be printed out or saved in files that can be imported by other programs.