I don't know if the question about Windows and blastclust ever got answered. I have been running blastclust on Windows XP since Sept 2005, I downloaded the blast_2.2.12 package of Windows executables. I have a nbci.ini file in my C:\WINDOWS directory which contains 2 line: [NCBI] Data=C:\... (the path to the blast \data directory) I also updated my Environment Variable PATH to include the blast \bin directory. Like Dan, whom replied with other info, I use a Perl script to invoke blastclust from a system() call within a Perl script. However you should be able to test calling blastclust from the DOS command line.