file('/var/lock/Application.lock', 'w').write(str(os.getpid()))
>
>  Which to be honest appears to run just fine, when I look in that file it
> always contains the correct process ID, for instance, 3419 or something like
> that.
>
I honestly doubt that. The I/O is buffered and you need to flush()/close()
the file. Otherwise data may not get written until python interpreter exits.

      if file('/proc/%s/cmdline' % pid).read().endswith('python'):
>
>             os.system('kill %s' % pid)
>
Are you sure about endswith('python')?
Maybe you need find('python') != -1.

-- 
Regards,
Arvind
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